Glucagon-like-peptide-2 (glp-2) analogues

ABSTRACT

GLP-2 analogues are disclosed which comprise one of more substitutions as compared to [hGly2]GLP-2 and which improved biological activity in vivo and/or improved chemical stability, e.g., as assessed in in vitro stability assays. More particularly, preferred GLP-2 analogues disclosed herein comprise substitutions at one or more of positions 8, 16, 24 and/or 28 of the wild-type GLP-2 sequence, optionally in combination with further substitutions at position 2 (as mentioned in the introduction) and one or more of positions 3, 5, 7, 10 and 11, and/or a deletion of one or more of amino acids 31 to 33 and/or the addition of a N-terminal or C-terminal stabilizing peptide sequence. The analogues are particularly useful for the prophylaxis or treatment of stomach and bowel-related disorders and for ameliorating side effects of chemotherapy. Also disclosed are methods and kits for selecting a patient from populations suited for treatment with GLP-2 analogues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/643,233, filed Dec. 21, 2009, which is a continuation of U.S.application Ser. No. 11/595,496, filed Nov. 9, 2006, which is acontinuation in part of U.S. application Ser. No. 11/429,168, filed May4, 2006, which, in turn, claims benefit from U.S. ProvisionalApplication No. 60/678,066, filed May 4, 2005, each of which is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to glucagon-like-peptide-2 (GLP-2)analogues and their medical use, for example in the prophylaxis ortreatment of stomach and bowel-related disorders and for amelioratingside effects of chemotherapy and radiation therapy.

BACKGROUND OF THE INVENTION

GLP-2 is a 33-amino-acid peptide released from the posttranslationalprocessing of proglucagon in the enteroendocrine L cells of theintestine and in specific regions of the brainstem. It is co-secretedtogether with glucagon-like peptide 1 (GLP-1), oxyntomodulin andglicentin, in response to nutrient ingestion.

GLP-2 induces significant growth of the small intestinal mucosalepithelium via the stimulation of stem cell proliferation in the cryptsand inhibition of apoptosis on the villi (Drucker et al. Proc Natl AcadSci USA. 1996, 93:7911-6). GLP-2 also inhibits gastric emptying andgastric acid secretion (Wojdemann et al. J Clin Endocrinol Metab. 1999,84:2513-7), enhances intestinal barrier function (Benjamin et al. Gut.2000, 47:112-9.), stimulates intestinal hexose transport via theupregulation of glucose transporters (Cheeseman, Am J Physiol. 1997,R1965-71), and increases intestinal blood flow (Guan et al.Gastroenterology. 2003, 125, 136-47).

GLP-2 binds to a single G protein-coupled receptor belonging to theclass II glucagon secretin family (1). The GLP-2 receptor has only beenlocalized in the small intestine, colon and stomach, sites that areknown to be responsive to GLP-2 (Yusta et al. Gastroenterology. 2000,119: 744-55). However, the target cell for GLP-2 receptor stimulation inthe gastrointestinal tract remains unclear and the downstreamintracellular mediators coupled to the GLP-2 receptor are poorlyunderstood.

The demonstrated specific and beneficial effects of GLP-2 in the smallintestine has raised much interest as to the use of GLP-2 in thetreatment of intestinal disease or injury (Sinclair and Drucker,Physiology 2005: 357-65). Furthermore GLP-2 has been shown to prevent orreduce mucosal epithelial damage in a wide number of preclinical modelsof gut injury, including chemotherapy-induced mucositis,ischemia-reperfusion injury, dextran sulfate-induced colitis and geneticmodels of inflammatory bowel disease (Sinclair and Drucker, Physiology2005:357-65).

GLP-2 is secreted as a 33 amino acid peptide with the following sequenceH-His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH (SEQ ID NO:50). It is rapidly cleaved at the Alanine (A) inposition 2 of the NH₂ terminus to the inactive human GLP-2 (3-33) by theenzyme DPP IV. This rapid enzymatic degradation of GLP-2(1-33), inaddition to renal clearance result in a half life of about 7 minutes forthe peptide (Tavares et al., Am. J. Physiol. Endocrinol. Metab.278:E134-E139, 2000).

In U.S. Pat. No. 5,994,500 (Drucker et al.) describes antagonists of theGLP-2 and their effects on the growth of gastrointestinal tissue. It issuggested that the antagonists are formulated as pharmaceuticals to beused in the treatment of hyperplasia or to induce hypoplasia. In U.S.Pat. No. 5,994,500 the structure of mammalian GLP-2 has been altered bymutations, such as substitutions and deletions.

U.S. Pat. No. 6,184,208, U.S. Pat. No. 5,789,379, and U.S. Pat. No.6,184,201 disclose GLP-2 analogues and their medical uses. The analoguesare all obtained by substitutions and/or deletions of the human GLP-2.

DaCambra et al. (Biochemistry 2000, 39, 8888-8894) describe thestructural determinants for activity of GLP-2. Examples of suchdeterminants are Phe6 and Thr5, which are referred to as crucial forGLP-2 receptor binding and activation.

In WO 97/39031 the GLP-2 analogue, [Gly2]GLP-2 (SEQ ID NO:54) isdisclosed. Here the alanine in position 2 has been replaced with glycineto make the peptide resistant to DPP IV cleavage. The replacement ofalanine is shown to increase the stability and potency of the peptide.The patent application describes how the GLP-2 analogue may be usedagainst diseases associated with inflammation and destruction of theintestinal epithelial mucosa. These include massive small intestineresection, inflammatory bowel disease, chemotherapy induced mucositisand ischemic injury.

WO 02/066511 describes GLP-2 analogues having an extended half-life invivo and their use as medicaments in the treatment of gastrointestinaldisorders, such as inflammatory bowel diseases.

WO 01/41779 describes the use of h[Gly2]GLP-2 (SEQ ID NO:54) as apretreatment for inhibiting chemotherapy induced apoptosis and promotingcell survival.

All references cited herein are expressly incorporated by reference intheir entirety.

The use of GLP-2 or analogues of GLP-2 in the treatment of variousdiseases has been proposed by many scientists. However, there is still aneed for improved and stable GLP-2 analogues.

SUMMARY OF THE INVENTION

Broadly, the present invention concerns GLP-2 analogues which compriseone of more substitutions as compared to wild-type GLP-2 and which mayhave the property of an improved biological activity in vivo and/orimproved chemical stability, e.g. as assessed in in vitro stabilityassays. More particularly, preferred GLP-2 analogues of the presentinvention comprise substitutions at one or more of positions 8, 16, 24and/or 28 of the wild-type GLP-2 sequence, optionally in combinationwith further substitutions at position 2 (as mentioned in theintroduction) and one or more of positions 3, 5, 7, 10 and 11, and/or adeletion of one or more of amino acids corresponding to positions 31 to33 of the wild-type GLP-2 sequence and/or the addition of a N-terminalor C-terminal stabilizing peptide sequence. As well as providing GLP-2analogues that may have improved chemical stability and/or biologicalactivity, the present invention also relates to providing compounds thathave preferential intestinal growth promoting activity in the smallintestine compared to the colon and vice versa, in particular byincluding modification at one or more of positions Asp3 and/or Ser 8and/or Asn16 and/or Asn24 and/or Gln28 of wild-type GLP-2.

Accordingly, in one aspect, the present invention provides a GLP-2analogue which is represented by general Formula I (SEQ ID NO:51):

R¹—Z¹-His-X2-X3-Gly-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-Ala-X19-X20-X21-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²—R²

wherein:

R¹ is hydrogen, C₁₋₄ alkyl (e.g. methyl), acetyl, formyl, benzoyl ortrifluoroacetyl

X2 is Gly, Ala or Sar

X3 is Glu or Asp

X5 is Ser or Thr

X6 is Phe or Pro

X7 is Ser or Thr

X8 is Asp or Ser

X9 is Glu or Asp

X10 is Met, Leu, Nle or an oxidatively stable Met-replacement amino acid

X11 is Asn, Ala, Lys or Ser

X12 is Thr or Lys

X13 is Ile, Glu or Gln

X14 is Leu, Met or Nle

X15 is Asp or Glu

X16 is Asn or Ala

X17 is Leu or Glu

X18 is Ala or Aib

X19 is Ala or Thr

X20 is Arg or Lys

X21 is Asp or Ile

X24 is Asn, Ala or Glu

X28 is Gln, Ala or Asn

X31 is Pro, Ile or deleted

X32 is Thr or deleted

X33 is Asp, Asn or deleted

R² is NH₂ or OH;

Z¹ and Z² are independently absent or a peptide sequence of 3-20 aminoacid units selected from the group consisting of Ala, Leu, Ser, Thr,Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met and Orn;

wherein the GLP-2 analogue comprises one or more of substitutionsselected from X8 is Ser and/or X16 is Ala and/or X24 is Ala and/or X28is Ala;or a pharmaceutically acceptable salt or derivative thereof.

In a further embodiment, the present invention provides a GLP-2 analoguerepresented by general Formula II (SEQ ID NO:52):

R¹—Z¹-His-Gly-X3-Gly-X5-Phe-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-Ala-X19-Arg-Asp-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²—R²

wherein:

R¹ is hydrogen, C₁₋₄ alkyl (e.g. methyl), acetyl, formyl, benzoyl ortrifluoroacetyl

X3 is Glu or Asp

X5 is Ser or Thr

X7 is Ser or Thr

X8 is Asp or Ser

X9 is Glu or Asp

X10 is Met, Leu, Nle or an oxidatively stable Met-replacement amino acid

X11 is Asn, Ala, Lys or Ser

X12 is Thr or Lys

X13 is Ile, Glu or Gln

X14 is Leu, Met or Nle

X15 is Asp or Glu

X16 is Asn or Ala

X17 is Leu or Glu

X19 is Ala or Thr

X24 is Asn or Ala

X28 is Gln, Ala or Asn

X31 is Pro, Ile or deleted

X32 is Thr or deleted

X33 is Asp or deleted

R² is NH₂ or OH;

Z¹ and Z² are independently absent or a peptide sequence of 3-20 aminoacid units selected from the group consisting of Ala, Leu, Ser, Thr,Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met and Orn;

wherein the GLP-2 analogue comprises one or more of substitutionsselected fromX8 is Ser and/or X16 is Ala and/or X24 is Ala and/or X28 is Ala;or a pharmaceutically acceptable salt or derivative thereof.

In a further embodiment, the present invention provides a GLP-2 analoguerepresented by general Formula III (SEQ ID NO:53):

R¹—Z¹-His-Gly-X3-Gly-X5-Phe-X7-X8-Glu-X10-X11-Thr-Ile-Leu-Asp-X16-Leu-Ala-Ala-Arg-Asp-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²—R²

wherein:

R¹ is hydrogen, C₁₋₄ alkyl (e.g. methyl), acetyl, formyl, benzoyl ortrifluoroacetyl

X3 is Glu or Asp

X5 is Ser or Thr

X7 is Ser or Thr

X8 is Asp or Ser

X10 is Met, Leu, Nle, or an oxidatively stable Met-replacement aminoacid

X11 is Asn, Ala, Lys or Ser

X24 is Asn or Ala

X28 is Gln or Ala

X31 is Pro or deleted

X32 is Thr or deleted

X33 is Asp or deleted

R² is NH₂ or OH;

Z¹ and Z² are independently absent or a peptide sequence of 3-20 aminoacid units selected from the group consisting of Ala, Leu, Ser, Thr,Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met and Orn

wherein the GLP-2 analogue comprises one or more of substitutionsselected fromX8 is Ser and/or X16 is Ala and/or X24 is Ala and/or X28 is Ala;or a pharmaceutically acceptable salt or derivative thereof. Where X16is not Ala, it will be Asn.

In certain embodiments, when Z¹ is present R¹ may be H, and when Z² ispresent R² may be OH.

In some embodiments of the present invention, the GLP-2 analogue has atleast 60% amino acid sequence identity to wild-type GLP-2 (1-33) havingthe sequence set out in the introduction of the application (SEQ IDNO:50), more preferably at least 63% sequence identity, more preferablyat least 66% sequence identity and still more preferably at least 69%sequence identity.

“Percent (%) amino acid sequence identity” with respect to the GLP-2polypeptide sequences is defined as the percentage of amino acidresidues in a candidate sequence that are identical with the amino acidresidues in the wild-type GLP-2 sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity, and not considering any conservative substitutions aspart of the sequence identity. Sequence alignment can be carried out bythe skilled person using techniques well known in the art for exampleusing publicly available software such as BLAST, BLAST2 or Alignsoftware, see:

-   Altschul et al (Methods in Enzymology, 266:460-480 (1996);-   http://blast.wustl/edu/blast/README.html) or Pearson et al    (Genomics, 46, 24, 36, 1997) and-   http://molbiol.soton.ac.uk/compute/aliqn.html for the Align program.

The percentage sequence identities used herein and in accordance withthe present invention are determined using these programs with theirdefault settings. More generally, the skilled person can readilydetermine appropriate parameters for determining alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared.

In some preferred embodiments, the GLP-2 peptide analogues representedby Formula I, II or III comprise substitutions at more than one ofpositions X8, X16, X24 and/or X28 and/or a combination of thesesubstitutions with other substitutions, preferably those at positionsX3, X5, X7, X10 and/or X11.

Examples of combinations of X8, X16, X24 and/or X28 substitutions thatfall within Formulae I to III include:

Ser8, Ala16

Ser8, Ala24

Ser8, Ala28

Ala16, Ala24

Ala16, Ala28

Ala24, Ala28

Ser8, Ala16, Ala24

Ser8, Ala16, Ala28

Ser8, Ala24, Ala28

Ala16, Ala24, Ala28

Ser8, Ala16, Ala24, Ala28

Examples of substitutions at positions X3, X5, X7, X10 and/or X11 thatfall within Formulae I to III and may be combined with a substitution atone or more of positions X8, X16, X24 and/or X28 include:

Glu3, Leu10, Ala11,24

Glu3, Thr5, Leu10, Ser11, Ala16,24,28

Glu3, Thr5, Leu10, Lys11, Ala16,24,28

Glu3, Thr5, Ser8, Leu10, Lys11, Ala16,24,28

Glu3, Thr5, Ser8,11, Leu10, Ala16,24,28

Glu3, Thr5, Ser8,11, Leu10, Ala16,24,28

Glu3, Ser8,11, Leu10, Ala16,24,28

Glu3, Leu10, Ser11, Ala16,24,28

Glu3, Leu10, Lys11, Ala16,24,28

Glu3, Thr5, Leu10, Ala11,16,24,28

Glu3, Thr5, Leu10, Ala11,16,24,28, Ile21

Glu3, Thr5, Ser8, Leu10, Ala11,16,24,28

Glu3, Ser8, Leu10, Ala11,16,24,28

Glu3, Leu10, Ala11,16,24,28

Thr7, Leu10, Ala11, 24

Thr7, Leu10, Lys11, Ala24

Thr7, Leu10, Ser11, Ala24

Thr7, Leu10, Ser8,11, Ala24

Thr7, Ser8, Leu10, Ala11,24

Thr7, Ser8, Leu10, Lys11, Ala24

Ser8, Leu10, Ala11,24

Leu10, Ala24

Leu10, Ala11, Ala24

Leu10, Ala11,24,28

Leu10, Ala11,16,24,28

Leu10, Lys11, Ala24

Leu10, Ser11, Ala24

Leu10, Ser8,11, Ala24

or a deletion at one or more of positions X31-X33 in combination with anabove mentioned change at position 8, 16, 24 and/or 28.

Specific examples of the GLP-2 compounds of the present invention areset out in the detailed description below.

As well as providing GLP-2 analogues that may have improved chemicalstability and biological activity, the present invention also relates toproviding compounds that have preferential growth promoting activity inthe small intestine compared to the colon and vice versa. In particular,the experiments described herein show that substitution at positionsAsp3 and/or Ser 8 and/or Asn16 and/or Gln28 of wild-type GLP-2 provide apreferential increase of the small intestine weight when administered totest animals compared to the increase in colon mass. These findings meanthat the exemplified compounds may be useful for treating conditionswhere it is advantageous to have an increased growth promoting effect inthe small intestine, while having a lesser effect on the colon, and viceversa.

Thus, compounds that are preferred for causing growth of the smallintestine typically comprise one or more substitutions at positions 3,8, 16 and/or 28 of wild-type GLP-2. Such compounds may selectively causegrowth of the small intestine rather than the colon. They may thereforebe used for conditions affecting or related to the small intestine.

Preferably, such small intestine-selective compounds comprisesubstitutions at more than one of positions X3, X7, X16, X24, X28, X31,X32 and/or X33. Thus, the small-intestine-selective compounds maycomprise more than one of the substitutions X3 is Glu, X7 is Ser, X16 isAla, X24 is Ala, X28 is Ala, X31 is Ile, X32 is Thr and X33 is Asp. Theamino acid residues in positions X31, X32 and X33 may optionally bedeleted.

Exemplified compounds preferentially stimulating epithelial growth inthe small intestine include 1809 (SEQ ID NO:1), 1818 (SEQ ID NO:8), 1819(SEQ ID NO:9), 1820 (SEQ ID NO:10), 1826 (SEQ ID NO:16), 1827 (SEQ IDNO:17), 1844 (SEQ ID NO:32), 1845 (SEQ ID NO:33), 1846 (SEQ ID NO:34),1848 (SEQ ID NO:36), 1849 (SEQ ID NO:37), 1850 (SEQ ID NO:38), 1851 (SEQID NO:39), 1852 (SEQ ID NO:40), 1853 (SEQ ID NO:41), 1855 (SEQ IDNO:42), 1857 (SEQ ID NO:45), 1858 (SEQ ID NO:46), 1859 (SEQ ID NO:47)(see Table 1).

On the other hand, compounds of the present invention that do not havethese modifications, e.g., which comprise one or more substitutions atpositions 10, 11 and/or 24, may be preferred for inducing preferentialgrowth of the colon rather than the small intestine. They may thereforebe used for treatment of conditions affecting or related to the colon.

Such colon-selective compounds may comprise more than one of thesubstitutions at positions X3, X8 and/or X24. For example, they maycomprises more than one substitution selected from X3 is Asp, X8 is Aspand X24 is Ala. The amino acid residues in positions X31, X32 and X33may optionally be deleted.

Exemplified compounds preferentially stimulating epithelial growth inthe colon include 1830 (SEQ ID NO:20), 1831 (SEQ ID NO:21), 1835 (SEQ IDNO:25), 1836 (SEQ ID NO:26), 1839 (SEQ ID NO:27), 1840 (SEQ ID NO:28),1841 (SEQ ID NO:29), and 1843 (SEQ ID NO:31).

Exemplified compounds without preferential growth in small intestine orcolon: [Gly2]GLP-2 (i.e., reference molecule), 1559 (SEQ ID NO:54), 1821(SEQ ID NO:11), 1822 (SEQ ID NO:12), 1823 (SEQ ID NO:13), 1825 (SEQ IDNO:15), 1828 (SEQ ID NO:18), 1829 (SEQ ID NO:19), 1832 (SEQ ID NO:22),1833 (SEQ ID NO:23), 1834 (SEQ ID NO:24), 1842 (SEQ ID NO:30), 1854 (SEQID NO:42).

The compounds of the invention also have increased chemical stability,e.g., against acid hydrolysis, oxidation and deamidation. It is believedthat substitutions at positions X3 and/or X33 may improve stability toacid hydrolysis. Substitution at position X10 may improve oxidativestability. Substitution at one or more of positions X11, X16 and/or X24may increase stability against deamidation. The GLP-2 analogue of theinvention may therefore exhibit enhanced stability towards degradationin acidic solution, towards deamidation, and/or towards oxidativedegradation, relative to Gly2-GLP-2.

Preferably, the GLP-2 analogue maintains an observed purity of at least(40%, 50%, 60%, 70%, 80%, or 90%, 95%, or 99% relative to the initialpurity in at least one of the degradation tests described in Example 7below. Additionally or alternatively, it may maintain an observed purityof at least 60% relative to initial purity in a solution of HCl 0.1 Mafter 12 days. Additionally or alternatively it may maintain an observedpurity of at least 70% relative to initial purity in a solution ofNH4HCO3 0.1 M after 6 days.

In a further aspect, the present invention provides a compositioncomprising a GLP-2 analogue as defined herein, or a salt or derivativethereof, in admixture with a carrier. In preferred embodiments, thecomposition is a pharmaceutically acceptable composition and the carrieris a pharmaceutically acceptable carrier. The GLP-2 peptide analogue maybe a pharmaceutically acceptable acid addition salt of the GLP-2analogue.

In a further aspect, the present invention provides a GLP-2 analogue asdefined herein, or a salt thereof, for use in therapy.

In a further aspect, the present invention provides use of a GLP-2analogue, or a salt or derivative thereof for the preparation of amedicament for the treatment and/or prevention of stomach andbowel-related disorders, such as the treatment of neonatals withcompromised intestine function, osteoporosis, and DPP-IV(dipeptidylpeptidase-IV) mediated conditions. By way of example, thestomach and bowel-related disorders include ulcers, gastritis, digestiondisorders, malabsorption syndromes, short-gut syndrome, cul-de-sacsyndrome, inflammatory bowel disease, celiac sprue (for example arisingfrom gluten induced enteropathy or celiac disease), tropical sprue,hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn'sdisease), ulcerative colitis, irritable bowel syndrome associated withdiarrhea, small intestine damage and short bowel syndrome.

Other conditions that may be treated with the GLP-2 analogues of theinvention, or for which the GLP-2 analogues may be usefulprophylactically or therapeutically, include radiation enteritis,infectious or post-infectious enteritis, and small intestinal damage dueto toxic or other chemotherapeutic agents. This may requireadministration of the GLP-2 analogue prior to, concurrently with orfollowing a course of chemotherapy or radiation therapy in order toreduce side effects of chemotherapy such as diarrhea, abdominal crampingand vomiting, and reduce the consequent structural and functional damageof the intestinal epithelium resulting from the chemotherapy orradiation therapy.

The invention therefore also provides a therapeutic kit comprising acancer chemotherapy drug and a GLP-2 analogue of the present invention,each optionally in combination with a pharmaceutically acceptablecarrier. The two therapeutic agents may be packaged separately (e.g. inseparate vials) for separate administration, or may be provided in thesame composition. Thus the invention further provides a pharmaceuticalcomposition comprising a cancer chemotherapy drug and a GLP-2 analogueof the present invention in combination with a pharmaceuticallyacceptable carrier.

For patients having gastrointestinal mucosal neoplasia, or an increasedrisk of gastrointestinal mucosal neoplasia, it may be desirable toselect a compound so as to reduce or abrogate the risk of reduced sideeffects such as stimulation or aggravation of gastrointestinal mucosalneoplasia. For example, when selecting a compound for treating a patientwith colon neoplasia (whether benign or malignant), or at risk ofdeveloping colon neoplasia, it may be more appropriate to select acompound which is selective for the small intestine over the colon thana non-selective compound or a compound which is selective for the colonover the small intestine.

In other aspects, the present invention provides the use of the GLP-2analogues for the preparation of a medicament for the treatment and/orprevention of malnutrition, for example conditions such as the wastingsyndrome cachexia and anorexia.

In a further aspect, the present invention provides a nucleic acidmolecule comprising a nucleic acid sequence encoding a GLP-2 analogue ofas defined herein.

In further aspects, the present invention provides an expression vectorcomprising the above nucleic acid sequence, optionally in combinationwith sequences to direct its expression, and host cells transformed withthe expression vectors. Preferably the host cells are capable ofexpressing and secreting the GLP-2 analogue. In a still further aspect,the present invention provides a method of producing the GLP-2 analogue,the method comprising culturing the host cells under conditions suitablefor expressing the GLP-2 analogue and purifying the GLP-2 analogue thusproduced.

The invention further provides a nucleic acid of the invention, anexpression vector of the invention, or a host cell capable of expressingand secreting a GLP-2 analogue of the invention, for use in therapy. Itwill be understood that the nucleic acid, expression vector and hostcells may be used for treatment of any of the disorders described hereinwhich may be treated with the GLP-2 analogues themselves. References toa therapeutic composition comprising a GLP-2 analogue of the invention,or administration of a GLP-2 analogue of the invention, should thereforebe construed to encompass administration of a nucleic acid, expressionvector or host cell of the invention except where the context demandsotherwise.

In a further aspect, the present invention provides a method of treatinga stomach and bowel-related disorder in a patient in need thereof byadministering an effective amount a GLP-2 analogue as defined herein, ora salt or derivative thereof, or a nucleic acid, expression vector orhost cell of the invention. Examples of stomach and bowel-relateddisorders are provided above.

In a further aspect, the present invention provides a method of treatingor preventing a side effect of chemotherapy or radiation therapy in apatient in need thereof, the method comprising administering aneffective amount a GLP-2 analogue as defined herein, or a salt orderivative thereof, or a nucleic acid, expression vector or host cell ofthe invention.

In a further aspect, the present invention provides a method of treatingor preventing malnutrition, for example conditions such as the wastingsyndrome cachexia and anorexia, in a patient in need thereof, the methodcomprising administering an effective amount a GLP-2 analogue as definedherein, or a salt or derivative thereof, or a nucleic acid, expressionvector or host cell of the invention.

The invention also features a method of selecting a patient for GLP-2analogue therapy. The method includes determining whether the patienthas a stomach or bowel disorder and determining whether the patient hasor is at increased risk of having abnormal tissue growth, where nothaving or not being at increased risk of having the abnormal tissuegrowth indicates the patient for GLP-2 analogue therapy. The method mayfurther include administering a GLP-2 analogue to the patient (e.g., inan amount effective to treat the patient for the stomach or boweldisorder). The GLP-2 analogue may be any GLP-2 analogue described hereinsuch as an analogue of general Formula I, for example, the GLP-2analogue may include a plurality of substitutions of the amino acidsequence of wild-type GLP-2 (1-33) at positions selected from the groupconsisting of X3, X7, X16, X24, X28, X31, X32, and X33. Alternatively,the GLP-2 analogue may include at least one substitution selected fromthe group consisting of X3 is Glu, X7 is Ser, X16 is Ala, X24 is Ala,X28 is Ala, X31 is Ile, X32 is Thr, and X33 is Asp, and a deletion ofzero, one, two, or three of the amino acid residues in positions X31,X32, and X33, or a pharmaceutically acceptable salt or derivativethereof. The GLP-2 analogue may be selected from the group consisting ofcompound numbers 1827 (SEQ ID NO:17), 1844 (SEQ ID NO:32), 1845 (SEQ IDNO:33), 1846 (SEQ ID NO:34), 1848 (SEQ ID NO:36), 1849 (SEQ ID NO:37),1850 (SEQ ID NO:38), 1851 (SEQ ID NO:39), 1852 (SEQ ID NO:40), 1855 (SEQID NO:43), 1857 (SEQ ID NO:45), 1858 (SEQ ID NO:46), and 1859 (SEQ IDNO:47), or a pharmaceutically acceptable salt or derivative thereof. Theabnormal tissue growth may be caused by a neoplasm (e.g., gastricmucosal neoplasm, colonic mucosal neoplasm, adenocarcinoma, coloncancer, colorectal cancer, small intestine cancer, and stomach cancer).The stomach or bowel disorder may be selected from the group consistingof an ulcer, a digestion disorder, a malabsorption syndrome, short-gutsyndrome, cul-de-sac syndrome, inflammatory bowel disease, celiac sprue,tropical sprue, hypogammaglobulinemic sprue, enteritis, regionalenteritis (Crohn's disease), ulcerative colitis, small intestine damage,and short bowel syndrome.

The invention also features a kit including a GLP-2 analogue representedby general Formula I and instructions for administering the GLP-2analogue to a patient not having or not at an increased risk ofdeveloping abnormal tissue growth. The GLP-2 analogue may include aplurality of substitutions of the amino acid sequence of wild-type GLP-2(1-33) at positions selected from the group consisting of X3, X7, X16,X24, X28, X31, X32, and X33. The GLP-2 analogue may include at least onesubstitution selected from the group consisting of X3 is Glu, X7 is Ser,X16 is Ala, X24 is Ala, X28 is Ala, X31 is Ile, X32 is Thr, and X33 isAsp, and a deletion of zero, one, two, or three of the amino acidresidues in positions X31, X32, and X33, or a pharmaceuticallyacceptable salt or derivative thereof. The GLP-2 analogue may beselected from the group consisting of compound numbers 1827 (SEQ IDNO:17), 1844 (SEQ ID NO:32), 1845 (SEQ ID NO:33), 1846 (SEQ ID NO:34),1848 (SEQ ID NO:36), 1849 (SEQ ID NO:37), 1850 (SEQ ID NO:38), 1851 (SEQID NO:39), 1852 (SEQ ID NO:40), 1855 (SEQ ID NO:43), 1857 (SEQ IDNO:45), 1858 (SEQ ID NO:46), and 1859 (SEQ ID NO:47), or apharmaceutically acceptable salt or derivative thereof. The GLP-2analogue may be provided in a pharmaceutically acceptable carrier. Theabnormal tissue growth may be caused by a neoplasm. The instructions mayfurther indicate that the GLP-2 analogue is to be administered for thetreatment of a stomach or bowel disorder. The instructions may furtherindicate that the GLP-2 analogue is to be administered for the treatmentof any condition selected from the group consisting of an ulcer, adigestion disorder, a malabsorption syndrome, short-gut syndrome,cul-de-sac syndrome, inflammatory bowel disease, celiac sprue, tropicalsprue, hypogammaglobulinemic sprue, enteritis, regional enteritis(Crohn's disease), ulcerative colitis, small intestine damage, and shortbowel syndrome.

Embodiments of the present invention will now be described in moredetail by way of examples and not limitation with reference to theaccompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a set of graphs showing full dose-response data of four smallintestine selective compounds (compound Nos. 1846 (SEQ ID NO:34), 1855(SEQ ID NO:43), 1848 (SEQ ID NO:36), and 1858 (SEQ ID NO:46) on smallintestinal (SI) mass in C57BL mice. Compounds were administered bysubcutaneous injection b.i.d. for three days at the following doses: 0(vehicle), 5, 15, 45, 135, 405 nmol/kg (n=6/dose group). Responses ateach dose level were compared with responses obtained at the same doselevel in pair-treated mice treated with the non-selective referencecompound [Gly2]GLP-2 (SEQ ID NO:54). To correct for changes in bodyweight (BW), SI mass was expressed relative to BW (SI-BW ratio) and thegrowth response at each dose level was normalized to the responseobserved with the non-selective reference compound [Gly2]GLP-2 (SEQ IDNO:54). Results demonstrated that within the dose range 5-405 nmol/kg,the dose-response relationships of the small intestine selectivecompounds 1846 (SEQ ID NO:34), 1855 (SEQ ID NO:43), 1848 (SEQ ID NO:36),and 1858 (SEQ ID NO:46) were significantly different from thenon-selective reference compound [Gly2]GLP-2 (SEQ ID NO:54) (p<0.05 intwo-way ANOVA) and all four compounds stimulated small intestinal growthwith maximal responses that were siginificant greater than [Gly2]GLP-2(SEQ ID NO:54). Values are means±SEM. *: P<0.05 relative to theequimolar dose of [Gly2]GLP-2 (SEQ ID NO:54).

FIG. 2 is a set of graphs showing full dose-response data of four smallintestine selective compounds (compound nos. 1846 (SEQ ID NO:34), 1855(SEQ ID NO:43), 1848 (SEQ ID NO:36), 1858 (SEQ ID NO:46)) on the SmallIntestine (SI)-to-Colon Sensitivity Index mice relative to thenon-selective reference compound [Gly2]GLP-2 (SEQ ID NO:54). Compoundswere administered by subcutaneous injection b.i.d. for three days inC57BL mice at the following doses: 0 (vehicle), 5, 15, 45, 135, 405nmol/kg (n=6/dose group). Responses at each dose level were comparedwith responses obtained at the same dose level in pair-treated micetreated with the non-selective reference compound [Gly2]GLP-2 (SEQ IDNO:54). The SI-Colon Sensitivity Index was calculated as SI-massrelative to colon mass and the Sensitivity Index at each dose level wasnormalized to the response observed with the non-selective referencecompound [Gly2]GLP-2 (SEQ ID NO:54). Results demonstrated that withinthe dose range 5-405 nmol/kg, the dose-response relationships of thesmall intestine selective compounds 1857 (SEQ ID NO:45) and 1820 (SEQ IDNO:10) were significantly different from the non-selective referencecompound [Gly2]GLP-2 (SEQ ID NO:54) (p<0.05 in two-way ANOVA) andcompounds 1846 (SEQ ID NO:34), 1855 (SEQ ID NO:43), and 1848 (SEQ IDNO:36) demonstrated increased maximal small intestinal sensitivityrelative to [Gly2]GLP-2. Values are means±SEM. *: P<0.05 relative to theequimolar dose of [Gly2]GLP-2.

FIG. 3 is a set of graphs showing full dose-response data of two smallintestine selective compounds (compound nos. 1857 (SEQ ID NO:45), 1849(SEQ ID NO:37), and 1820 (SEQ ID NO:10) on small intestinal (SI) mass inC57BL mice. Compounds were administered by subcutaneous injection b.i.d.for three days at the following doses: 0 (vehicle), 5, 15, 45, 135, 405nmol/kg (n=6/dose group). Responses at each dose level were comparedwith responses obtained at the same dose level in pair-treated micetreated with the non-selective reference compound [Gly2]GLP-2. Tocorrect for changes in body weight (BW), SI mass was expressed relativeto BW (SI-BW ratio) and the growth response at each dose level wasnormalized to the response observed with the non-selective referencecompound [Gly2]GLP-2 (SEQ ID NO:54). Results demonstrated that withinthe dose range 5-405 nmol/kg, the dose-response relationships of thesmall intestine selective compounds 1857 (SEQ ID NO:45), 1849 (SEQ IDNO:37), and 1820 (SEQ ID NO:10) were significantly different from thenon-selective reference compound [Gly2]GLP-2 (SEQ ID NO:54) (p<0.05 intwo-way ANOVA) and both compounds stimulated small intestinal growthwith maximal responses that were significantly greater than [Gly2]GLP-2(SEQ ID NO:54). Values are means±SEM. *: P<0.05 relative to theequimolar dose of [Gly2]GLP-2 (SEQ ID NO:54).

FIG. 4 is a set of graphs showing full dose-response data of three smallintestine selective compounds (compound nos. 1857 (SEQ ID NO:45), 1820(SEQ ID NO:10), and 1849 (SEQ ID NO:37)) on the Small Intestine(SI)-to-Colon Sensitivity Index mice relative to the non-selectivereference compound [Gly2]GLP-2 (SEQ ID NO:54). Compounds wereadministered by subcutaneous injection b.i.d. for three days in C57BLmice at the following doses: 0 (vehicle), 5, 15, 45, 135, 405 nmol/kg(n=6/dose group). Responses at each dose level were compared withresponses obtained at the same dose level in pair-treated mice treatedwith the non-selective reference compound [Gly2]GLP-2 (SEQ ID NO:54).The SI-Colon Sensitivity Index was calculated as SI-mass relative tocolon mass and the Sensitivity Index at each dose level was normalizedto the response observed with the non-selective reference compound[Gly2]GLP-2 (SEQ ID NO:54). Results demonstrated that within the doserange 5-405 nmol/kg, the dose-response relationships of the smallintestine selective compounds 1857 (SEQ ID NO:45), 1820 (SEQ ID NO:10),and 1849 (SEQ ID NO:37) were significantly different from thenon-selective reference compound [Gly2]GLP-2 (SEQ ID NO:54) (p<0.05 intwo-way ANOVA) and all three compounds demonstrated increased maximalsmall intestinal sensitivity relative to [Gly2]GLP-2 (SEQ ID NO:54).Values are means±SEM. *: P<0.05 relative to the equimolar dose of[Gly2]GLP-2 (SEQ ID NO:54).

FIG. 5 shows examples of full dose-response data of a non-selectivecompound ([Gly2]GLP-2 (SEQ ID NO:54)), and a small intestine selectivecompound (Compound 1848 (SEQ ID NO:36)), on small intestine, colon andstomach mass. Compounds were administered by subcutaneous injectionb.i.d. for three days in C57BL mice at the following doses: 0 (vehicle),5, 15, 45, 135, 405 nmol/kg (n=6/dose group). Results demonstrated thatwithin the dose range 5-405 nmol/kg, [Gly2]GLP-2 (SEQ ID NO:54) produceddose-dependent growth stimulation in small intestine, colon and stomach,while Compound 1848 (SEQ ID NO:36) only produced dose-dependent growthstimulation in the small intestine. Values are means±SEM. *: P<0.05relative to the vehicle.

FIG. 6 is a set of graphs showing the effect of compound 1846 (SEQ IDNO:34) (5, 15, 45, 135, 405 and 810 nmol/kg, s.c., b.i.d.; n=6/dosegroup) on A) small intestine-to-body weight ratio (SI-BW), and B) smallintestinal length (SI length) in mice treated with the cytostatic drug5-Fluorouracil (5-FU). Compound 1846 (SEQ ID NO:34) was administered for3 days prior to and for 4 days together with 5-FU, 50 mg/kg, i.p. daily.For reference, [Gly2]GLP-2 (SEQ ID NO:54) (405 nmol/kg) was given to onegroup of animals. Results from control animals treated with vehicle(PBS) or 5-FU alone are also shown. Compound 1846 (SEQ ID NO:34)prevented 5-FU-induced small intestine atrophy (reduced organ mass andshortening) in C57BL mice. Values are means±SEM. * P<0.05, relative to5-FU.

FIG. 7 is a set of graphs showing the effect of compound 1848 (SEQ IDNO:36) (5, 15, 45, 135, 405 and 810 nmol/kg, s.c., b.i.d.; n=6/dosegroup) on A) small intestine-to-body weight ratio (SI-BW), and B) smallintestinal length (SI length) in mice treated with the cytostatic drug5-Fluorouracil (5-FU). Compound 1848 (SEQ ID NO:36) was administered for3 days prior to and for 4 days together with 5-FU, 50 mg/kg, i.p. daily.For reference, [Gly2]GLP-2 (SEQ ID NO:54) (405 nmol/kg) was given to onegroup of animals. Results from control animals treated with vehicle(PBS) or 5-FU alone are also shown. Compound 1848 (SEQ ID NO:36)prevented 5-FU-induced small intestine atrophy (reduced organ mass andshortening) in C57BL mice and high doses of compound 1848 (SEQ ID NO:36)were more efficacious than [Gly2]GLP-2. Values are means±SEM. * P<0.05,relative to 5-FU. # P<0.05, relative to [Gly2]GLP-2.

FIG. 8 is a set of graphs showing the effect of compound 1855 (SEQ IDNO:43) (5, 15, 45, 135, and 405 nmol/kg, s.c., b.i.d.; n=6/dose group)on A) small intestine-to-body weight ratio (SI-BW), and B) smallintestinal length (SI length) in mice treated with the cytostatic drug5-Fluorouracil (5-FU). Compound 1855 (SEQ ID NO:43) was administered for3 days prior to and for 4 days together with 5-FU, 50 mg/kg, i.p. daily.For reference, [Gly2]GLP-2 (405 nmol/kg) was given to one group ofanimals. Results from control animals treated with vehicle (PBS) or 5-FUalone are also shown. Compound 1855 (SEQ ID NO:43) prevented5-FU-induced small intestine atrophy (reduced organ mass and shortening)in C57BL mice and the highest dose of compound 1855 (SEQ ID NO:43) wasmore efficacious than the equimolar dose of [Gly2]GLP-2 (SEQ ID NO:54).Values are means±SEM. * P<0.05, relative to 5-FU. # P<0.05, relative to[Gly2]GLP-2 (SEQ ID NO:54).

FIG. 9 is a set of graphs showing the effect of compound 1857 (SEQ IDNO:45) (5, 15, 45, 135, and 405 nmol/kg, s.c., b.i.d.; n=6/dose group)on A) small intestine-to-body weight ratio (SI-BW), and B) smallintestinal length (SI length) in mice treated with the cytostatic drug5-Fluorouracil (5-FU). Compound 1857 (SEQ ID NO:45) was administered for3 days prior to and for 4 days together with 5-FU, 50 mg/kg, i.p. daily.For reference, [Gly2]GLP-2 (SEQ ID NO:54) (405 nmol/kg) was given to onegroup of animals. Results from control animals treated with vehicle(PBS) or 5-FU alone are also shown. Compound 1857 (SEQ ID NO:45)prevented 5-FU-induced small intestine atrophy (reduced organ mass andshortening) in C57BL mice and the highest doses of compound 1855 (SEQ IDNO:43) were more efficacious than a high dose of [Gly2]GLP-2 (SEQ IDNO:54). Values are means±SEM. * P<0.05, relative to 5-FU. # P<0.05,relative to [Gly2]GLP-2 (SEQ ID NO:54).

FIG. 10 is a set of graphs showing effects of compound 1846 (SEQ IDNO:34) (16, 80 and 400 nmol/kg/d; s.c., n=6/dose group) in maleSpague-Dawley rats treated with the cytostatic drug 5-Fluorouracil(5-FU). Compound 1846 (SEQ ID NO:34) was administered for 3 days priorto and for 4 days together with 5-FU (75 mg/kg, i.p. daily). Resultswere compared with responses in control animals treated with vehicle(saline) or 5-FU alone. Saline and 5-FU controls are also shown.Compound 1846 (SEQ ID NO:34) prevented 5-FU-induced small intestineatrophy (reduced small intestinal mass and intestinal shortening) inrats. Values are means±SEM. * P<0.05, relative to 5-FU alone.

FIG. 11 is a set of graphs showing the therapeutic effect of compound1846 (SEQ ID NO:34) on diarrhea induced by treatment with the cytostaticdrug 5-fluorouracil (5-FU). Rats were treated with 5-FU for 4 days with75 mg/kg once daily (days 1-4); (n=40 rats). Half of the animalsreceived additional treatment with compound 1846 (SEQ ID NO:34) (400nmol/kg s.c. once daily) during the last 3 days prior to (days −3 to −1)and during 4 days of 5-FU treatment (days 0 to 3). Rats were observedtwice daily (morning and evening) to determine whether the animal haddiarrhea and the severity of the diarrhea was scored according to thisscale: (0) no diarrhea; (1) mild—fecal staining around the anus; (2)moderate—fecal staining on the hind limbs and tail; and (3) severe—fecalstaining on the front limbs and abdomen. On day 5, about 70% of SpragueDawley rats that received 5-FU alone had developed diarrhea (FIG. 11A)while only 30% of the rats that were co-treated with compound 1846 (SEQID NO:34) developed diarrhea (FIG. 11B). These results indicate thatcompound 1846 (SEQ ID NO:34) effectively prevents injury of the smallintestine and thus the development of diarrhea during cytostatictreatment with 5-FU.

FIG. 12 is a set of graphs showing the effect of compound 1846 (SEQ IDNO:34) on epithelial crypt-villus height (FIG. 12A) and muscularthickness (FIG. 12B) in the jejunum (i.e., mid section of the smallintestine). Sprague Dawley rats were treated intravenously with compound1846 (SEQ ID NO:34) (0.62, 3.41 or 6.2 mg/kg once daily) for 5 days(n=6/dose group). Then the animals were sacrified and small intestinalbiopsies (1 cm) were collected 25 cm distal from the pyloric sphincter.The biopsies were fixed, embedded in paraffin, sectioned and stainedwith haematoxylin and eosin. Stained sections were examined under amicroscope and crypt depth, villus height, crypt-villus length andmuscularis thickness measured. As illustrated compound 1846 (SEQ IDNO:34) produced dose-dependent increases in crypt-villus length inintestinal epithelium from the jejunum, but had no effect on themuscular thickness of the jejunum. These results suggest that compound1846 (SEQ ID NO:34) primarily exerts its proliferative actions in thesmall intestine through stimulation of small epithelial growth. Valuesare means±SEM. * P<0.05, relative to vehicle controls (0 mg/kg/d).

FIG. 13 is a graph illustrating the effect of compound 1848 (SEQ IDNO:36) on small intestinal ulcers induced by indomethacin. Male SpragueDawley rats were treated with vehicle (saline; group 1) or indomethacin(7 mg/kg s.c., once daily for two days; groups 2-7). Indomethacin wasgiven alone (group 2) or in combination with prednisolone (10 mg/kgs.c.; group 3) or with 8, 40, or 200 nmol/kg compound 1848 (SEQ IDNO:36) s.c. once daily (groups 4-6). Finally, one group of rats wastreated with combination treatment of prednisolone (10 mg/kg s.c.) andcompound 1848 (SEQ ID NO:36), 200 nmol/kg s.c. once daily (group 7).Treatment with compound 1848 (SEQ ID NO:36) was initiated 4 days priorto indomethacin and continued during the two days of indomethacintreatment. On day 3, rats were sacrificed and upon necropsy, the smallintestine was gently flushed with 10% formalin and filled with formalinfor a period of 5 minutes, after which the intestine was cut open alongthe antimesenteric margin and suspended on a polypropylene plate. Anyremaining intestinal contents were carefully removed with a pair oftweezers. After fixation for another 24 hours at room temperature, thetissue was rinsed in Milli-Q water and surface-stained for 20 minuteswith Alcian Green 3BX (Chroma) prepared as a 0.5% solution in 1% aceticacid (Bie & Berntsen). After removal of excess staining solution withMilli-Q water the tissue preparation was transferred to 70% alcohol andanalyzed using a stereomicroscope at low magnification (×7). Starting atthe pylorus, the small intestine was scanned and the shape (circular vs.linear) and size (circular ulcers: diameter, linear ulcers:length×width) of all ulcers was measured using a standard ruler(resolution: 0.5 mm). An ulcer was defined as an area, which lackedepithelial surface. In healing ulcers only the area that still lackedepithelial surface was regarded an ulcer even if the villus structurewas still missing in a larger area. All analysis was performed in ablinded manner. As illustrated in FIG. 13, indomethacin caused a stronginduction of small intestinal ulcers (total small intestinalulceration=333±21 mm²). Treatment with prednisolone caused a significantreduction in the extent of ulceration by app. 29%. Treatment withcompound 1848 (SEQ ID NO:36) prevented indomethacin-induced ulcerationin a dose-dependent fashion and at the highest dose the total ulcerationwas reduced by almost 50% (178±17 mm²). This maximal response tocompound 1846 (SEQ ID NO:34) was greater than the effect of prednisoloneand addition of prednisolone in combination with high dose compound 1848(SEQ ID NO:36) did not produced any additional effect. These resultsindicate that compound 1848 (SEQ ID NO:36) effectively preventsindomethacin-induced ulceration in the small intestine. Values aremeans±SEM. * P<0.05 vs. indomethacin (group 2). ^(#)P<0.05 vs.prednisolone (group 3).

FIG. 14 is a set of graphs showing the effect of compound 1848 (SEQ IDNO:36) on small intestinal content of TNF-alpha in rats withindomethacin-induced inflammation in the small intestine. Male SpragueDawley rats were treated with vehicle (saline; group 1) or indomethacin(7 mg/kg s.c., once daily for two days; groups 2-7). Indomethacin wasgiven alone (group 2) or in combination with prednisolone (10 mg/kgs.c.; group 3) or with 8, 40, or 200 nmol/kg compound 1848 (SEQ IDNO:36) s.c. once daily (groups 4-6). Finally, one group of rats wastreated with combination treatment of prednisolone (10 mg/kg s.c.) andcompound 1848 (SEQ ID NO:36), 200 nmol/kg s.c. once daily (group 7).Treatment with compound 1848 (SEQ ID NO:36) was initiated 4 days priorto indomethacin and continued during the two days of indomethacintreatment. On day 3, rats were sacrificed and upon necropsy, the smallintestine was divided into three segments of equal length correspondingto 1) duodenum and proximal jejunum, 2) mid and distal jejunum and 3)ileum. Samples were immediately snap-frozen in liquid N₂ and stored at−80° C. until analysis. Homogenization and extraction of cellularprotein from small intestinal segments was performed according to thefollowing procedure: Tissue segments were weighed and added a volume of1.5 ml per gram tissue ice-cold (4° C.) extraction buffer (10 mMTris-HCl (Sigma) and 1 mM EDTA (J. T. Baker), (pH 7.6) with 0.05% sodiumazide (Fluka), 1% Tween-80 (Fluka), 2 mM phenylmethylsulfonyl fluoride(PMSF, Fluka) and 1 μg/ml of each of the protease inhibitors aprotinin,leupeptin and pepstatin A (Roche Diagnostics)). The tissue was cut intosmall bits with a pair of scissors and homogenized three times for 20seconds (IKA UltraTurrax T25 homogenizer, Janke & Kunkel) at 9500 rpmwith intermittent cooling on ice for 30 seconds. The homogenate wascentrifuged at 20.000×g for 30 minutes at 4° C., the supernatant wascollected and stored at −20° C. until analysis for TNF-α smallintestinal protein extractions were analyzed for TNF-α using acommercially available ELISA kit according to the manufacturer'sinstructions (Rat Tumor Necrosis Factor-α UltraSensitive ELISA kit,Biosource International Inc.). The assay has a detection limit of 0.7pg/ml. Protein extractions were analyzed for total protein content usinga commercially available assay (DC protein assay, Bio-Rad LaboratoriesLtd.). Small intestinal tissue levels of TNF-α were expressed relativeto total protein. Compound 1848 (SEQ ID NO:36) significantly decreasedsmall intestinal tissue levels of the proinflammatory cytokine TNF-α andthis effect was most marked in the proximal segment (first ⅓^(rd) ofsmall intestine). Compound 1848 (SEQ ID NO:36) was more efficacious thanprednisolone in the proximal and mid segments (second ⅓^(rd) of smallintestine). Interestingly, compound 1848 (SEQ ID NO:36) suppressedinflammation in the small intestine more effectively than prednisone,and prednisone had no additive effect when given in combination withcompound 1848 (SEQ ID NO:36). These results suggest that compound 1848(SEQ ID NO:36) has a marked anti-inflammatory potential on diseasesprocesses that affect the small intestine. Values are means±SEM. *P<0.05 vs. indomethacin (group 2).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless specified otherwise, the following definitions are provided forspecific terms, which are used in the above written description.

Throughout the description and claims the conventional one-letter andthree-letter codes for natural amino acids are used as well as generallyaccepted three letter codes for other α-amino acids, such as sarcosin(Sar), norleucine (Nle) and α-aminoisobutyric acid (Aib). All amino acidresidues in peptides of the invention are preferably of theL-configuration, However, D-configuration amino acids may also bepresent.

Preferred compounds of the present invention have at least one GLP-2biological activity, in particular in causing growth of the intestine.This can be assessed in in vivo assays, for example as described in theexamples, in which the mass of the intestine, or a portion thereof isdetermined after a test animal has been treated or exposed to a GLP-2analogue.

The GLP-2 analogues of the present invention have one or more amino acidsubstitutions, deletions, inversions, or additions compared with nativeGLP-2 and as defined above. This definition also includes the synonymterms GLP-2 mimetics and/or GLP-2 agonists. Further, the analogue of thepresent invention may additionally have chemical modification of one ormore of its amino acid side groups, α-carbon atoms, terminal aminogroup, or terminal carboxylic acid group. A chemical modificationincludes, but is not limited to, adding chemical moieties, creating newbonds, and removing chemical moieties. Modifications at amino acid sidegroups include, without limitation, acylation of lysine ε-amino groups,N-alkylation of arginine, histidine, or lysine, alkylation of glutamicor aspartic carboxylic acid groups, and deamidation of glutamine orasparagine. Modifications of the terminal amino include, withoutlimitation, the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acylmodifications. Modifications of the terminal carboxy group include,without limitation, the amide, lower alkyl amide, dialkyl amide, andlower alkyl ester modifications. Preferably herein lower alkyl is C₁-C₄alkyl. Furthermore, one or more side groups, or terminal groups, may beprotected by protective groups known to the ordinarily-skilled peptidechemist. The α-carbon of an amino acid may be mono- or di-methylated.

Where they are present, oxidatively stable Met-replacement amino acidmeans one which is selected among the group consisting of Met(O)(methionine sulfoxide), Met(O)₂ (methionine sulfone), Val, Ile, Asn, Glx(Glu or Gln), Tyr, Phe, Trp and preferably Leu, Nle, Ala, Ser, and Gly.

By a peptide with “enhanced stability” is meant a peptide (e.g., a GLP-2peptide described herein) having a increased resistance to degradation(e.g., at least 5%, 10%, 25%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%) lessas compared a reference peptide (e.g., Gly2-GLP-2 (SEQ ID NO:54)) undera given set of conditions over a given time period (e.g., at least 1, 6,or 12, or 1, 2, 3, 5, 6, 8, 10, 12, 15, 20, 30, 45, or 60 days).Alternatively or additionally, stability may be assayed by comparing thepercent purity in a sample subjected to a set of conditions a comparedto the same sample not subjected to the set of conditions. Anyconditions described herein or known in the art may be used to testenhanced stability. In particular embodiments, stability is tested underacidic conditions (e.g., at least 0.01, 0.1, 0.2, 0.4, 0.5, 1.0, 2.0, or5.0 M HCl), basic conditions (e.g., at least 0.01, 0.1, 0.2, 0.4, 0.5M1.0, 2.0, or 5.0 M NaOH), oxidative stress (e.g., at least 0.01% 0.05%,0.1%, 0.2%, 0.5%, 1.0%, 1.5%, 2.0%, 5%, or 10%, 20%, or 30% H₂O₂), ordeamindation conditions (e.g., at least 0.01, 0.1, 0.2, 0.4, 0.5, 1.0,2.0, or 5.0 M NH₄HCO₃). Conditions also include (either alone or incombination) with the conditions listed such as increased temperature(e.g., at 30, 35, 37, 40, 45, 50, 60, 70, 80, 100, 150° C.). Exemplaryconditions are described in Table 9 below.

It should be understood that the peptides of the invention might also beprovided in the form of a salt or other derivative. Salts includepharmaceutically acceptable salts such as acid addition salts and basicsalts. Examples of acid addition salts include hydrochloride salts,citrate salts and acetate salts. Examples of basic salts include saltswhere the cation is selected from alkali metals, such as sodium andpotassium, alkaline earth metals, such as calcium, and ammonium ions⁺N(R³)₃(R⁴), where R³ and R⁴ independently designates optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionallysubstituted aryl, or optionally substituted heteroaryl. Other examplesof pharmaceutically acceptable salts are described in “Remington'sPharmaceutical Sciences,” 17th edition. Ed. Alfonso R. Gennaro (Ed.),Mark Publishing Company, Easton, Pa., U.S.A., 1985 and more recenteditions, and in the Encyclopedia of Pharmaceutical Technology.

Other derivatives of the GLP-2 analogues of the invention includecoordination complexes with metal ions such as Mn²⁺ and Zn²⁺ esters suchas in vivo hydrolysable esters, free acids or bases, hydrates, prodrugsor lipids. Esters can be formed between hydroxyl or carboxylic acidgroups present in the compound and an appropriate carboxylic acid oralcohol reaction partner, using techniques well known in the art.Derivatives which act as prodrugs of the compounds are convertible invivo or in vitro into one of the parent compounds. Typically, at leastone of the biological activities of compound will be reduced in theprodrug form of the compound, and can be activated by conversion of theprodrug to release the compound or a metabolite of it. Examples ofprodrugs include the use of protecting groups which may be removed insitu releasing active compound or serve to inhibit clearance of the drugin vivo.

When present, Z¹ and Z² each independently represent a peptide sequenceof 3-20 or 4-20 amino acid residues, e.g. in the range of 4-15, morepreferably in the range of 4-10 in particular in the range of 4-7 aminoacid residues, e.g., of 4, 5, 6 or 7 amino acid residues, such as 6amino acid residues. Each of the amino acid residues in the peptidesequences Z may independently be selected from Ala, Leu, Ser, Thr, Tyr,Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn. Preferably, the amino acidresidues are selected from Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg,His, Orn, and Met, as well as amino acids falling within formula I asdefined in WO01/04156, e.g., Dbu (2,4 diaminobutyric acid) or Dpr(2,3-diaminopropanoic acid), more preferably from Glu, Lys, and Met,especially Lys. The above-mentioned amino acids may have either D- orL-configuration, but preferably the above-mentioned amino acids have anL-configuration. Particularly preferred sequences Z are sequences offour, five or six consecutive lysine residues, and particularly sixconsecutive lysine residues. Exemplary sequences Z are shown in WO01/04156.

In certain embodiments, Z¹ is absent. In such cases, Z² may be eitherpresent or absent.

The present invention includes the following peptides further describedin the experimental section below.

Reference GLP-2 analogue 1559 H-[Gly2]hGLP-2-OH (SEQ ID NO: 54)H-HGDGSFSDEMNTILDNLAARDFINWLIQTKITD-OHExamples of GLP-2 analogues of the present invention1809 [Gly2, Glu3, Thr5, Leu10, Ala11, 16, 24, 28]hGLP-2-(Lys)₆-NH₂(SEQ ID NO: 1) HGEGTFSDELATILDALAARDFIAWLIATKITDK₆-NH₂1810 [Gly2, Glu3, Thr5, Leu10, Ala11,16, 24,28, Ile21]hGLP-2(1-30)-(Lys)₆-NH₂ (SEQ ID NO: 2)HGEGTFSDELATILDALAARIFIAWLIATK₆-NH₂1811 [Gly2, Pro6, Leu10, Ala11, 16,24, 28] hGLP-2-NH₂ (SEQ ID NO: 3)HGDGSPSDELATILDALAARDFIAWLIATKITD-NH₂1812 [Gly2, Glu3, Leu10, Ala11, 24]hGLP-2-NH₂ (SEQ ID NO: 4)HGEGSFSDELATILDNLAARDFIAWLIQTKITD-NH₂1813 [Gly2, Leu10, Ala11, 16, 24, 28]hGLP-2-NH₂ (SEQ ID NO: 5)H-HGDGSFSDELATILDALAARDFIAWLIATKITD-NH₂1814 [Gly2, Leu10, Ala11, 24, 28]hGLP-2-NH₂ (SEQ ID NO: 6)H-HGDGSFSDELATILDNLAARDFIAWLIATKITD-NH₂1815 [Gly2, Glu3, Leu10, Ala11, 16, 24, 28] hGLP-2-NH₂ (SEQ ID NO: 7)H-HGEGSFSDELATILDALAARDFIAWLIATKITD-NH₂1818 [Gly2, Ser8, Leu10, Ala11, 24]hGLP- 2(1-30)-K₆-NH₂ (SEQ ID NO: 8)H-HGDGSFSSELATILDNLAARDFIAWLIQTK₆-NH₂1819 [Gly2, Leu10, Ser11, Ala24]hGLP- 2(1-30)-K₆-NH₂ (SEQ ID NO: 9)H-HGDGSFSDELSTILDNLAARDFIAWLIQTK₆-NH₂1820 [Gly2, Thr7, Ser8, Leu10, Ala11, 24]hGLP-2(1-30)-K₆-NH₂(SEQ ID NO: 10) H-HGDGSFTSELATILDNLAARDFIAWLIQTK₆-NH₂1821 [Gly2, Leu10, Lys11, Ala24]hGLP-2(1-30)- K₆-NH₂ (SEQ ID NO: 11)H-HGDGSFSDELKTILDNLAARDFIAWLIQTK₆-NH₂1822 [Gly2, Thr7, Leu10, Lys11, Ala24]hGLP- 2(1-30)-K₆-NH₂(SEQ ID NO: 12) H-HGDGSFTDELKTILDNLAARDFIAWLIQTK₆-NH₂1823 [Gly2, Thr7, Ser8, Leu10, Lys11, Ala24]hGLP-2(1-30)-K₆-NH₂(SEQ ID NO: 13) H-HGDGSFTSELKTILDNLAARDFIAWLIQTK₆-NH₂1824 [Gly2, Thr7, Leu10, Ala11, 24]hGLP- 2(1-30)-K₆-NH₂ (SEQ ID NO: 14)H-HGDGSFTDELATILDNLAARDFIAWLIQTK₆-NH₂1825 [Gly2, Ser8, Leu10, Ala11, 24]hGLP- 2(1-30)-NH₂ (SEQ ID NO: 15)H-HGDGSFSSELATILDNLAARDFIAWLIQTK-NH₂1826 [Gly2, Leu10, Ala24]hGLP-2-K₆-NH₂ (SEQ ID NO: 16)H-HGDGSFSDELNTILDNLAARDFIAWLIQTKITDK₆-NH₂1827 [Gly2, Thr7, Leu10, Ser11, Ala24]hGLP- 2(1-30)-K₆-NH₂(SEQ ID NO: 17) H-HGDGSFTDELSTILDNLAARDFIAWLIQTK₆-NH₂1828 [Gly2, Thr7, Leu10, Ser8, 11, Ala24]hGLP- 2(1-30)-K₆-NH₂(SEQ ID NO: 18) H-HGDGSFTSELSTILDNLAARDFIAWLIQTK₆-NH₂1829 [Gly2, Leu10, Ser8, 11, Ala24]hGLP- 2(1-30)-K₆-NH₂ (SEQ ID NO: 19)H-HGDGSFSSELSTILDNLAARDFIAWLIQTK₆-NH₂1830 [Gly2, Leu10, Ser11, Ala24]hGLP- 2(1-30)-NH₂ (SEQ ID NO: 20)H-HGDGSFSDELSTILDNLAARDFIAWLIQTK-NH₂ 1831 [Gly2, Thr7, Leu10, Ser11,Ala24]hGLP-2(1-30)-NH₂ (SEQ ID NO: 21)H-HGDGSFTDELSTILDNLAARDFIAWLIQTK-NH₂ 1832 [Gly2, Thr7, Leu10, Ser8, 11,Ala24]hGLP-2(1-30)-NH₂ (SEQ ID NO: 22)H-HGDGSFTSELSTILDNLAARDFIAWLIQTK-NH₂1833 [Gly2, Leu10, Ser8, 11, Ala24]hGLP- 2(1-30)-NH₂ (SEQ ID NO: 23)H-HGDGSFSSELSTILDNLAARDFIAWLIQTK-NH₂1834 [Gly2, Thr7, Ser8, Leu10, Ala11, 24]hGLP-2(1-30)-NH₂(SEQ ID NO: 24) H-HGDGSFTSELATILDNLAARDFIAWLIQTK-NH₂1835 [Gly2, Leu10, Lys11, Ala24]hGLP- 2(1-30)-NH₂ (SEQ ID NO: 25)H-HGDGSFSDELKTILDNLAARDFIAWLIQTK-NH₂1836 [Gly2, Thr7, Leu10, Lys11, Ala24]hGLP- 2(1-30)-NH₂ (SEQ ID NO: 26)H-HGDGSFTDELKTILDNLAARDFIAWLIQTK-NH₂1839 [Leu10, Ala11, Ala24]hGLP-2 (1-33)-K₆-NH₂ (SEQ ID NO: 27)H-HGDGSFSDELATILDNLAARDFIAWLIQTKITDK₆-NH₂1840 [Leu10, Ala11, Ala24]hGLP-2 (1-33)-NH₂ (SEQ ID NO: 28)H-HGDGSFSDELATILDNLAARDFIAWLIQTKITD-NH₂1841 [Leu10, Ala11, Ala24]hGLP-2 (1-30)-NH₂ (SEQ ID NO: 29)H-HGDGSFSDELATILDNLAARDFIAWLIQTK-NH₂1842 [Thr7, Ser8, Leu10, Lys11, Ala24]hGLP- 2 (1-30)-NH₂ (SEQ ID NO: 30)H-HGDGSFTSELKTILDNLAARDFIAWLIQTK-NH₂1843 [Thr7, Leu10, Ala11, Ala24]hGLP-2 (1-30)-NH₂ (SEQ ID NO: 31)H-HGDGSFTDELATILDNLAARDFIAWLIQTK-NH₂1844 [Gly2, Glu3, Thr5, Ser8, 11, Leu10,Ala16, 24, 28]hGLP-2(1-33)-(Lys)₆-NH₂ (SEQ ID NO: 32)H-HGEGTFSSELSTILDALAARDFIAWLIATKITDK₆-NH₂1845 [Gly2, Glu3, Thr5, Leu10, Ser11,Ala16, 24, 28]hGLP-2(1-33)-(Lys)₆-NH₂ (SEQ ID NO: 33)H-HGEGTFSDELSTILDALAARDFIAWLIATKITDK₆-NH₂1846 [Gly2, Glu3, Ser8, 11, Leu10, Ala16, 24, 28]hGLP-2(1-33)-(Lys)₆-NH₂(SEQ ID NO: 34) H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK₆NH₂1847 [Gly2, Glu3, Leu10, Ser11, Ala16, 24, 28]hGLP-2(1-33)-(Lys)₆-NH₂(SEQ ID NO: 35) H-HGEGSFSDELSTILDALAARDFIAWLIATKITDK₆-NH₂1848 [Gly2, Glu3, Thr5, Ser8, Leu10, Ala11,16, 24, 28]hGLP-2(1-33)-(Lys)₆-NH₂ (SEQ ID NO: 36)H-HGEGTFSSELATILDALAARDFIAWLIATKITDK₆-NH₂1849 [Gly2, Glu3, Ser8, Leu10, Ala11, 16, 24, 28]hGLP-2(1-33)-(Lys)₆-NH₂(SEQ ID NO: 37) H-HGEGSFSSELATILDALAARDFIAWLIATKITDK₆-NH₂1850 [Gly2, Glu3, Leu10, Lys11, Ala16, 24, 28]hGLP-2(1-33)-(Lys)₆-NH₂(SEQ ID NO: 38) H-HGEGSFSDELKTILDALAARDFIAWLIATKITDK₆-NH₂1851 [Gly2, Glu3, Thr5, Leu10, Lys11, Ala16,24, 28]hGLP-2(1-33)-(Lys)₆-NH₂ (SEQ ID NO: 39)H-HGEGTFSDELKTILDALAARDFIAWLIATKITDK₆-NH₂1852 [Gly2, Glu3, Thr5, Ser8, Leu10, Lys11,Ala16, 24, 28]hGLP-2(1-33)-NH₂ (SEQ ID NO: 40)H-HGEGTFSSELKTILDALAARDFIAWLIATKITDK₆-NH₂1853 [Gly2, Glu3, Thr5, Ser8, 11, Leu10, Ala16, 24, 28]hGLP-2(1-33)-NH₂(SEQ ID NO: 41) H-HGEGTFSSELSTILDALAARDFIAWLIATKITD-NH₂1854 [Gly2, Glu3, Thr5, Leu10, Ser11, Ala16, 24, 28]hGLP-2(1-33)-NH₂(SEQ ID NO: 42) H-HGEGTFSDELSTILDALAARDFIAWLIATKITD-NH₂1855 [Gly2, Glu3, Thr5, Ser8, Leu10, Ala11, 16, 24, 28]hGLP-2(1-33)-NH₂(SEQ ID NO: 43) H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH₂1856 [Gly2, Glu3, Ser8, 11, Leu10, Ala16, 24, 28]hGLP-2(1-33)-NH₂(SEQ ID NO: 44) H-HGEGSFSDELSTILDALAARDFIAWLIATKITD-NH₂1857 [Gly2, Glu3, Leu10, Ser11, Ala16, 24, 28]hGLP-2(1-33)-NH₂(SEQ ID NO: 45) H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH₂1858 [Gly2, Glu3, Ser8, Leu10, Ala11, 16, 24, 28]hGLP-2(1-33)-NH₂(SEQ ID NO: 46) H-HGEGSFSSELATILDALAARDFIAWLIATKITD-NH₂1859 [Gly2, Glu3, Leu10, Lys11, Ala16, 24, 28]hGLP-2(1-33)-NH₂(SEQ ID NO: 47) H-HGEGSFSDELKTILDALAARDFIAWLIATKITD-NH₂1860 [Gly2, Glu3, Thr5, Leu10, Lys11, Ala16, 24, 28]hGLP-2(1-33)-NH₂(SEQ ID NO: 48) H-HGEGTFSDELKTILDALAARDFIAWLIATKITD-NH₂1861 [Gly2, Glu3, Thr5, Ser8, Leu10, Lys11,Ala16, 24, 28]hGLP-2(1-33)-NH₂ (SEQ ID NO: 49)HGEGTFSSELKTILDALAARDFIAWLIATKITD

Particularly preferred compounds of the present invention includecompounds 1834 (SEQ ID NO:24), 1846 (SEQ ID NO:34), 1847 (SEQ ID NO:35),1848 (SEQ ID NO:36), 1849 (SEQ ID NO:37), 1855 (SEQ ID NO:43), 1857 (SEQID NO:45), and 1858 (SEQ ID NO:46).

(SEQ ID NO: 24) 1834 H-HGDGSFTSELATILDNLAARDFIAWLIQTK-NH₂(SEQ ID NO: 34) 1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK₆NH₂(SEQ ID NO: 35) 1847 H-HGEGSFSDELSTILDALAARDFIAWLIATKITDK₆-NH₂(SEQ ID NO: 36) 1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDK₆-NH₂(SEQ ID NO: 37) 1849 H-HGEGSFSSELATILDALAARDFIAWLIATKITDK₆-NH₂(SEQ ID NO: 43) 1855 H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH₂(SEQ ID NO: 45) 1857 H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH₂(SEQ ID NO: 46) 1858 H-HGEGSFSSELATILDALAARDFIAWLIATKITD-NH₂

By “increased risk” is meant an elevated (e.g., 10%, 20%, 50%, 100%,200%, 500%, 1000% greater) risk of a patient developing a disease orcondition as compared to a control patient. For example, a person with amutation in a gene linked to colon cancer is at an increased risk ofdeveloping colon cancer as compared to a person without the samemutation.

Stability Studies

The skilled person will be able to design appropriate methods (e.g.quantitative methods) for detection of degradation products of GLP-2analogues, e.g. based on those described below. Degradation may occur asoxidation, hydrolysis and deamidation, depending on the identity andposition of the amino acids in any given GLP-2 analogue, and conditionsas pH, solution and temperature. The compounds can be ranked accordingto chemical stability, when the compounds are incubated under stressedconditions (i.e. conditions likely to cause degradation) andsubsequently analyzed for content of remaining intact peptide. Inaddition, the knowledge gained about major degradation products obtainedunder stressed conditions will be important for any later analyticalmethod development.

Quantitative Assays to Detect GLP Analogues

The skilled person will also be capable of designing methods (e.g.quantitative methods) for detection of GLP analogues in complexenvironments or solutions (e.g. plasma, urine, tissue homogenates, cellhomogenates, saliva or similar) to investigate the absorption,distribution, metabolism and excretion of the GLP analogues afteradministration to mammals or as part of functional studies of in vitrocell systems.

In one embodiment, a quantitative assay can be based on antibodiesraised against the GLP analogues or fragments thereof. The antibodiesobtained from the immunized animals can be used for quantitative assays.In one example a direct sandwich ELISA can be prepared using a firstantibody with affinity of one part of the molecule immobilized in amulti-well plate. The sample is then applied to the wells and the GLPanalogue is captured by the first antibody. The captured GLP analogue isthen recognized by a second antibody with affinity for another part ofthe GLP analogue. The second antibody can be labeled with an enzyme(horseradish peroxidase, alkaline phosphatase or beta-galactosidase) ora radioisotope. The amount of captured GLP analogue can then be detectedby addition of a colorimetric substrate or direct counting ofradio-emission or by scintillation. Alternatively, the amount ofcaptured GLP analogue can be detected indirectly by addition of alabeled antibody with affinity for the second antibody. Theconcentration in the sample can be estimated from the response obtainedfrom an external standard curve containing known amounts of GLPanalogue. Alternatively, the antibodies can be used to prepare a directcompetitive immuno assay, where an antibody specific for the GLPanalogue is immobilized on a multi-well plate and the sample incubatedin the wells with a predefined fixed concentration of labeled GLPanalogue. The label can be an enzyme, a fluorophore, a radioisotope orbiotin and detected using, for example, substrates (e.g. colorimetric,fluorometric or chemiluminiscent) specific for the enzymes,scintillation or avidin linked to an enzyme followed by detection asdescribed above. The amount of bound labeled GLP analogue can bedetected by an appropriate method and the concentration of GLP analoguepresent in the sample derived from the response obtained from anexternal standard curve as described above.

In another embodiment, a quantitative assay can be based on liquidchromatography tandem mass spectroscopy methodology. In such a set up,the response from a fragment specific for the GLP analogue to be studiedis monitored upon fragmentation of the parent compound induced bycollision with an inert gas (He or Ar). Prior to fragmentation thesample components can be separated by reversed phase chromatography orthe sample can be injected directly in the mass spectrometer. Ifsuitable the sample can be subjected to pretreatment (i.e., addition ofprotease inhibitors, protein precipitation, solid phase extraction,immuno-affinity extraction, etc. The concentration of GLP analoguepresent in the sample derived from the response obtained from anexternal standard curve as described above, potentially after correctionof the response using an internal standard similar to the GLP analogueto be studied.

Generation of Specific Antibodies

Specific antibodies against the GLP analogues or fragments thereof canbe induced in mammals and purified from the serum. The GLP analogues orfragments can either be used directly with an adjuvant to immunizerabbits, mice or other mammals, or the GLP analogues or fragmentsthereof can be chemically linked to a carrier molecule (i.e., keyholelimpet hemocyanin, ovalbumin, albumin etc.) and injected with anadjuvant. The injections can be repeated with 2-4 weeks intervals forextended periods to improve the affinity and selectivity of theantibodies. Polyclonal antibodies can be harvested directly from theserum. To obtain monoclonal antibodies, B cells isolated from immunizedanimals, preferably mice, should be fused with tumor cells to formantibody producing hybridomas. Screening and selection of theappropriate clones and antibodies can be performed using eitherimmobilized GLP analogues or peptides thereof followed by detection withlabeled anti-antibodies. Alternatively the screening and selection couldbe based on immobilized antibodies followed by detection with labeledGLP analogues or fragments thereof. In all cases, the label could be aradioisotope, an enzyme, a fluorophore or biotin and detected using, forexample, substrates (e.g. colorimetric, fluorometric orchemiluminiscent) specific for the enzymes, scintillation or avid inlinked to an enzyme followed by detection as described.

Synthesis of GLP-2 Analogues

It is preferred to synthesize the analogues of the invention by means ofsolid phase or liquid phase peptide synthesis. In this context,reference is given to WO 98/11125 and, amongst many others, Fields, G Bet al., 2002, “Principles and practice of solid-phase peptidesynthesis”. In: Synthetic Peptides (2nd Edition) and the Examplesherein.

Thus the GLP-2 analogues may be synthesized in a number of waysincluding for example, a method which comprises:

(a) synthesizing the peptide by means of solid phase or liquid phasepeptide synthesis and recovering the synthetic peptide thus obtained; or(b) when the peptide is constituted by naturally occurring amino acids,expressing a nucleic acid construct that encodes the peptide in a hostcell and recovering the expression product from the host cell culture;or(c) when the peptide is constituted by naturally occurring amino acids,effecting cell-free in vitro expression of a nucleic acid construct thatencodes the peptide and recovering the expression product; ora combination of methods of (a), (b), and (c) to obtain fragments of thepeptide, subsequently ligating the fragments to obtain the peptide, andrecovering the peptide.

Thus, for some analogues of the invention it may be advantageous toexploit genetic engineering techniques. This may be the case when thepeptide is sufficiently large (or produced as a fusion construct) andwhen the peptide only includes naturally occurring amino acids that canbe translated from RNA in living organisms.

For the purpose of recombinant gene technology nucleic acid fragmentsencoding the peptides of the invention are important chemical products.Hence, a further aspect of the present invention provides a nucleic acidmolecule comprising a nucleic acid sequence encoding a GLP-2 analogue ofthe invention, where the peptide preferably is comprised by naturallyoccurring amino acids. The nucleic acid fragments of the invention areeither DNA or RNA fragments.

The nucleic acid fragments of the invention will normally be inserted insuitable vectors to form cloning or expression vectors carrying thenucleic acid fragments of the invention; such novel vectors are alsopart of the invention. Details concerning the construction of thesevectors of the invention will be discussed in context of transformedcells and microorganisms below. The vectors can, depending on purposeand type of application, be in the form of plasmids, phages, cosmids,mini-chromosomes, or virus, but also naked DNA which is only expressedtransiently in certain cells is an important vector. Preferred cloningand expression vectors (plasmid vectors) of the invention are capable ofautonomous replication, thereby enabling high copy-numbers for thepurposes of high-level expression or high-level replication forsubsequent cloning.

The general outline of a vector of the invention comprises the followingfeatures in the direction and in operable linkage: a promoter fordriving expression of the nucleic acid fragment of the invention,optionally a nucleic acid sequence encoding a leader peptide enablingsecretion (to the extracellular phase or, where applicable, into theperiplasma) of or a leader peptide for multiple use e.g. combinedsecretion, purification tag and enzymatic trimming to correct peptide orintegration into the membrane of the polypeptide fragment, the nucleicacid fragment encoding the peptide of the invention, and optionally anucleic acid sequence encoding a terminator. When operating withexpression vectors in producer strains or cell-lines it is for thepurposes of genetic stability of the transformed cell preferred that thevector when introduced into a host cell is integrated in the host cellgenome.

The vectors of the invention are used to transform host cells to producethe modified peptide of the invention. Such transformed cells, which arealso part of the invention, can be cultured cells or cell lines used forpropagation of the nucleic acid fragments and vectors of the invention,or used for recombinant production of the peptides of the invention.

Preferred transformed cells of the invention are micro-organisms such asbacteria (such as the species Escherichia (e.g. E. coli), Bacillus (e.g.Bacillus subtilis), Salmonella, or Mycobacterium (preferablynon-pathogenic, e.g. M. bovis BCG)), yeasts (such as Saccharomycescerevisiae), and protozoans. Alternatively, the transformed cells arederived from a multicellular organism, i.e. it may be fungal cell, aninsect cell, a plant cell, or a mammalian cell. Also cells derived froma human being are interesting, cf. the discussion of cell lines andvectors below. For the purposes of cloning and/or optimised expressionit is preferred that the transformed cell is capable of replicating thenucleic acid fragment of the invention. Cells expressing the nucleicfragment are preferred useful embodiments of the invention; they can beused for small-scale or large-scale preparation of the peptides of theinvention.

When producing the peptide of the invention by means of transformedcells, it is convenient, although far from essential, that theexpression product is either exported out into the culture medium orcarried on the surface of the transformed cell.

When an effective producer cell has been identified it is preferred, onthe basis thereof, to establish a stable cell line which carries thevector of the invention and which expresses the nucleic acid fragmentencoding the peptide. Preferably, this stable cell line secretes orcarries the peptide of the invention, thereby facilitating purificationthereof.

In general, plasmid vectors containing replicon and control sequences,which are derived from species compatible with the host cell, are usedin connection with the hosts. The vector ordinarily carries areplication site, as well as marking sequences, which are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322 (but numerous other usefulplasmids exist) a plasmid derived from an E. coli species (see, e.g.,Bolivar et al., 1977). The pBR322 plasmid contains genes for ampicillinand tetracycline resistance and thus provides easy means for identifyingtransformed cells. The pBR plasmid, or other microbial plasmid or phagemust also contain, or be modified to contain promoters, which can beused by the prokaryotic microorganism for expression.

Those promoters most commonly used in prokaryotic recombinant DNAconstruction include the β-lactamase (penicillinase) and lactosepromoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel etal., 1979) and a tryptophan (trp) promoter system (Goeddel et al., 1979;EP 0 036 776 A). While these are the most commonly used, other microbialpromoters have been discovered and utilized, and details concerningtheir nucleotide sequences have been published, enabling a skilledworker to ligate them functionally with plasmid vectors (Siebwenlist etal., 1980).

In addition to prokaryotes, eukaryotic microbes, such as yeast culturesmay also be used, and also here the promoter should be capable ofdriving expression. Saccharomyces cerevisiae, or common baker's yeast isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomyces, the plasmid YRp7, for example, is commonly used(Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al.,1980). This plasmid already contains the trpl gene which provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan for example ATCC No. 44076 or PEP4-1 (Jones, 1977).The presence of the trpl lesion as a characteristic of the yeast hostcell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzman et al., 1980) or other glycolyticenzymes (Hess et al., 1968; Holland et al., 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination.

Other promoters, which have the additional advantage of transcriptioncontrolled by growth conditions are the promoter region for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, and the aforementionedglyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible formaltose and galactose utilization. Any plasmid vector containing ayeast-compatible promoter, origin of replication and terminationsequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate in culture (tissue culture) has become aroutine procedure in recent years (Tissue Culture, 1973). Examples ofsuch useful host cell lines are VERO and HeLa cells, Chinese hamsterovary (CHO) cell lines, and W138, BHK, COS-7 293, Spodoptera frugiperda(SF) cells (commercially available as complete expression systems fromi.a. Protein Sciences, 1000 Research Parkway, Meriden, Conn. 06450,U.S.A. and from Invitrogen), the D. melanogaster cell line S₂ availablefrom Invitrogen, PO Box 2312, 9704 CH Groningen, The Netherlands, andMDCK cell lines.

Expression vectors for such cells ordinarily include (if necessary) anorigin of replication, a promoter located in front of the gene to beexpressed, along with any necessary ribosome binding sites, RNA splicesites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment, which also contains the SV40 viral origin of replication(Fiers et al., 1978). Smaller or larger SV40 fragments may also be used,provided there is included the approximately 250 bp sequence extendingfrom the HindIII site toward the Bgll site located in the viral originof replication. Further, it is also possible, and often desirable, toutilize promoter or control sequences normally associated with thedesired gene sequence, provided such control sequences are compatiblewith the host cell systems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g. Polyoma, Adeno, VSV, BPV) or may be provided by thehost cell chromosomal replication mechanism. If the vector is integratedinto the host cell chromosome, the latter is often sufficient.

In order to obtain satisfactory yields in a recombinant productionprocess, it may be advantageous to prepare the analogues as fusionproteins, either by fusing the peptide to a fusion partner that canserve as an affinity tag (for ease of purification) and/or by havingmultiple repeats of the peptide. These methods require presence of asuitable cleavage site for a peptidase, but the skilled person will knowhow to tailor the underlying genetic constructs.

After recombinant preparation, the peptides of the invention can bepurified by methods generally known in the art, including multi-stepchromatography (e.g., ion-exchange, size-exclusion, and affinitychromatographic techniques).

Alternatively, peptides comprised of naturally occurring amino acids canbe prepared in vitro in cell free systems. This is especially expedientin cases where the peptides could be toxic for putative host cells.Thus, the present invention also contemplates use of cell-free in vitrotranslation/expression in order to prepare the peptides of theinvention. In this context, reference is made to commercially availablein vitro translation kits, materials, and technical documentation frome.g., Ambion Inc., 2130 Woodward, Austin, Tex. 78744-1832, USA.

Finally, the available methods can of course be combined so as toprepare, e.g., semi-synthetic analogues. In such a set up, peptidefragments are prepared using at least 2 separate steps or methods,followed by ligation of the fragments to obtain the final peptideproduct.

Pharmaceutical Compositions and Administration

The GLP-2 analogues of the present invention, or salts or derivativesthereof, may be formulated as pharmaceutical compositions prepared forstorage or administration, and which comprise a therapeuticallyeffective amount of a GLP-2 peptide of the present invention, or a saltor derivative thereof, in a pharmaceutically acceptable carrier.

The therapeutically effective amount of a compound of the presentinvention will depend on the route of administration, the type of mammalbeing treated, and the physical characteristics of the specific mammalunder consideration. These factors and their relationship to determiningthis amount are well known to skilled practitioners in the medical arts.This amount and the method of administration can be tailored to achieveoptimal efficacy so as to deliver the peptide to the large intestine,but will depend on such factors as weight, diet, concurrent medicationand other factors, well known those skilled in the medical arts.

It is within the invention to provide a pharmaceutical composition,wherein the GLP-2 analogue, or a salt thereof is present in an amounteffective to treat or prevent stomach and bowel-related disorders.

Pharmaceutically acceptable salts of the compounds of the inventionhaving an acidic moiety can be formed using organic and inorganic bases.Suitable salts formed with bases include metal salts, such as alkalimetal or alkaline earth metal salts, for example sodium, potassium, ormagnesium salts; ammonia salts and organic amine salts, such as thoseformed with morpholine, thiomorpholine, piperidine, pyrrolidine, amono-, di- or tri-lower alkylamine (e.g., ethyl-tert-butyl-, diethyl-,diisopropyl-, triethyl-, tributyl- or dimethylpropylamine), or a mono-,di- or trihydroxy lower alkylamine (e.g., mono-, di- ortriethanolamine). Internal salts also may be formed. Similarly, when acompound of the present invention contains a basic moiety, salts can beformed using organic and inorganic acids. For example, salts can beformed from the following acids: acetic, propionic, lactic, citric,tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic,hydrochloric, hydrobromic, phosphoric, nitric, sulfuric,methanesulfonic, napthalenesulfonic, benzenesulfonic, toluenesulfonic,and camphorsulfonic as well as other known pharmaceutically acceptableacids. Amino acid addition salts can also be formed with amino acidssuch as lysine, glycine, or phenylalanine.

As is apparent to one skilled in the medical art, a “therapeuticallyeffective amount” of the peptides or pharmaceutical compositions of thepresent invention will vary depending upon the age, weight and mammalianspecies treated, the particular compounds employed, the particular modeof administration and the desired effects and the therapeuticindication. Because these factors and their relationship to determiningthis amount are well known in the medical arts, the determination oftherapeutically effective dosage levels, the amount necessary to achievethe desired result of preventing and/or treating the intestine andstomach related diseases described herein, as well as other medicalindications disclosed herein, will be within the ambit of the skilledperson.

As used herein, “a therapeutically effective amount” is one whichreduces symptoms of a given condition or pathology, and preferably whichnormalizes physiological responses in an individual with the conditionor pathology. Reduction of symptoms or normalization of physiologicalresponses can be determined using methods routine in the art and mayvary with a given condition or pathology. In one aspect, atherapeutically effective amount of one or more GLP-2 analogues orpharmaceutical composition comprising the one or more GLP-2 analogues isan amount which restores a measurable physiological parameter tosubstantially the same value (preferably to within +30%, more preferablyto within +20%, and still more preferably, to within 10% of the value)of the parameter in an individual without the condition or pathology.

In one embodiment of the invention administration of the compounds orpharmaceutical composition of the present invention is commenced atlower dosage levels, with dosage levels being increased until thedesired effect of preventing/treating the relevant medical indication,such as intestine and stomach related diseases is achieved. This woulddefine a therapeutically effective amount. For the peptides of thepresent invention, alone or as part of a pharmaceutical composition,such doses may be between about 0.01 mg/kg and 100 mg/kg body weight,such as between about 0.01 mg/kg and 10 mg/kg body weight, for examplebetween 10-100 μg/kg body weight.

For therapeutic use, the chosen GLP-2 analogue is formulated with acarrier that is pharmaceutically acceptable and is appropriate fordelivering the peptide by the chosen route of administration. For thepurpose of the present invention, peripheral parenteral routes includeintravenous, intramuscular, subcutaneous, and intra peritoneal routes ofadministration. Certain compounds used in the present invention may alsobe amenable to administration by the oral, rectal, nasal, or lowerrespiratory routes. These are so-called non-parenteral routes. Thepresent pharmaceutical composition comprises a GLP-2 analogue of theinvention, or a salt or derivative thereof and a pharmaceuticallyacceptable carrier. Suitable pharmaceutically acceptable carriers arethose used conventionally with peptide-based drugs, such as diluents,excipients and the like. Pharmaceutically acceptable carriers fortherapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro edit. 1985). For example, sterile salineand phosphate-buffered saline at slightly acidic or physiological pH maybe used. pH buffering agents may be phosphate, citrate, acetate,tris/hydroxymethyl)aminomethane (TRIS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS),ammonium bicarbonate, diethanolamine, histidine, which is a preferredbuffer, arginine, lysine, or acetate or mixtures thereof. Preferredbuffer ranges are pH 4-8, pH 6.5-8, more preferably pH 7-7.5.Preservatives, such as para, meta, and ortho-cresol, methyl- andpropylparaben, phenol, benzyl alcohol, sodium benzoate, benzoic acid,benzyl-benzoate, sorbic acid, propanoic acid, esters of p-hydroxybenzoicacid may be provided in the pharmaceutical composition. Stabilizers,preventing oxidation, deamidation, isomerisation, racemisation,cyclisation, peptide hydrolysis, such as, e.g., ascorbic acid,methionine, tryptophane, EDTA, asparagine, lysine, arginine, glutamineand glycine may be provided in the pharmaceutical composition.Stabilizers, preventing aggregation, fibrillation, and precipitation,such as sodium dodecyl sulfate, polyethylene glycol, carboxymethylcellulose, cyclodextrine may be provided in the pharmaceuticalcomposition. Organic modifiers for solubilization or preventingaggregation, such as ethanol, acetic acid or acetate and salts thereofmay be provided in the pharmaceutical composition. Isotonicity makers,such as salts, e.g., sodium chloride or most preferred carbohydrates,e.g., dextrose, mannitol, lactose, trehalose, sucrose or mixturesthereof may be provided in the pharmaceutical composition.

Detergents, such as Tween 20, Tween 80, SDS, Poloxamers, e.g., PluronicF-68, Pluronic F-127, may be provided in the pharmaceutical composition.Dyes and even flavoring agents may be provided in the pharmaceuticalcomposition. In another embodiment, a pharmaceutically acceptable acidaddition salt of the GLP-2 peptide analogue is provided for. Suspendingagents may be used. Organic modifiers, such as ethanol,tertiary-buthanol, 2-propanol, ethanol, glycerol, Polyethylene glycolmay be provided in the pharmaceutical formulation for lyophilization ofa lyophilized product. Bulking agents and isotonicity makers such assalt e.g. sodium chloride, carbohydrates e.g. dextrose, mannitol,lactose, trehalose, sucrose or mixtures thereof, aminoacids e.g.glycine, glutamate, or excipients such as cystein, lecithin or humanserum albumin, or mixtures thereof may be provided in the pharmaceuticalcomposition for lyophilization.

The pharmaceutical compositions of the present invention may beformulated and used as tablets, capsules or elixirs for oraladministration; suppositories for rectal administration; preferablysterile solutions or sterile powder or suspensions for injectableadministration; and the like. The dose and method of administration canbe tailored to achieve optimal efficacy but will depend on such factorsas weight, diet, concurrent medication and other factors, which thoseskilled in the medical arts will recognize.

When administration is to be parenteral, such as intravenous andsubcutaneous, e.g., on a daily basis, injectable pharmaceuticalcompositions can be prepared in conventional forms, either as aqueoussolutions or suspensions; lyophilized, solid forms suitable forreconstitution immediately before use or suspension in liquid prior toinjection, or as emulsions.

Diluents for reconstitution of the lyophilized product may be a suitablebuffer from the list above, water, saline, dextrose, mannitol, lactose,trehalose, sucrose, lecithin, albumin, sodium glutamate, cysteinehydrochloride; or water for injection with addition of detergents, suchas Tween 20, Tween 80, poloxamers, e.g., pluronic F-68 or pluronicF-127, polyethylene glycol, and or with addition of preservatives suchas para-, meta-, and ortho-cresol, methyl- and propylparaben, phenol,benzyl alcohol, sodium benzoate, benzoic acid, benzyl-benzoate, sorbicacid, propanoic acid, esters of p-hydroxybenzoic acid, and or withaddition of an organic modifier such as ethanol, acitic acid, citricacid, lactic acid or salts thereof.

In addition, if desired, the injectable pharmaceutical compositions maycontain minor amounts of non-toxic auxiliary substances, such as wettingagents, or pH buffering agents. Absorption enhancing preparations (e.g.,liposomes, detergents and organic acids) may be utilized.

In one embodiment of the invention, the compounds are formulated foradministration by infusion, e.g., when used as liquid nutritionalsupplements for patients on total parenteral nutrition therapy (forexample neonatals, or patients suffering from cachexia or anorexia), orby injection, for example subcutaneously, intraperitoneal orintravenously, and are accordingly utilized as aqueous solutions insterile and pyrogen-free form and optionally buffered to physiologicallytolerable pH, e.g., a slightly acidic or physiological pH. Formulationfor intramuscular administration may be based on solutions orsuspensions in plant oil, e.g. canola oil, corn oil or soybean oil.These oil-based formulations may be stabilized by antioxidants e.g. BHA(butylated hydroxianisole) and BHT (butylated hydroxytoluene). Thus, thepresent peptide compounds may be administered in a vehicle, such asdistilled water or in saline, phosphate buffered saline, 5% dextrosesolutions or oils. The solubility of the GLP-2 analogue may be enhanced,if desired, by incorporating a solubility enhancer, such as detergentsand emulsifiers.

The aqueous carrier or vehicle can be supplemented for use asinjectables with an amount of gelatin that serves to depot the GLP-2analogue at or near the site of injection, for its slow release to thedesired site of action. Alternative gelling agents, such as hyaluronicacid, may also be useful as depot agents.

In one embodiment of the present invention the formulation comprises

a. L-histidine dissolved in water to obtain final concentrations of from0.5 mM to 300 mM, preferably from 3 to 200 mM, most preferably from 20to 100 mM;b. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM, mostpreferably from 100 mM to 230 mM; andc. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100 mM,most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/ml,most preferably from 10 to 30 mg/ml.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilized product.

In another embodiment of the present invention the formulation comprises

a. L-histidine dissolved in water to obtain final concentrations of from0.5 mM to 300 mM, preferably from 3 to 200 mM, most preferably from 20to 100 mM L-histidine;b. L-Arginine to obtain up to 200 mM, preferably from 0.5 to 100 mM,most preferably from 5 to 50 mM;c. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM, mostpreferably from 100 mM to 230 mM; andd. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100 mM,most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/ml, preferably from 5 to 50 mg/ml,most preferably from 10 to 30 mg/ml.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilized product.

In still another embodiment of the present invention the formulationcomprises

a. L-histidine dissolved in water to obtain final concentrations of upto 200 mM, preferably from 3 to 100 mM, most preferably from 5 to 50 mML-histidine;b. L-Arginine to obtain up to 200 mM, preferably from 0.5 to 100 mM,most preferably from 5 to 50 mM;c. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM, mostpreferably from 100 mM to 230 mM; andd. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100 mM,most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/ml, preferably from 5 to 50 mg/ml,most preferably from 10 to 30 mg/ml.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilized product.

In yet another embodiment of the present invention the formulationcomprises

a. L-histidine dissolved in water to obtain final concentrations of from0.5 to 300 mM, preferably from 3 to 200 mM, most preferably from 20 to100 mM L-histidine;b. L-Arginine to obtain up to 200 mM, preferably from 0.5 to 100 mM,most preferably from 5 to 50 mM;c. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM, mostpreferably from 100 mM to 230 mM; andd. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100 mM,most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/mL,most preferably from 10 to 30 mg/ml.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilized product.

In yet another embodiment of the present invention the formulationcomprises

a. L-histidine dissolved in water to obtain final concentrations of fromup to 200 mM, preferably from 3 to 100 mM, most preferably from 5 to 50mM L-histidine;b. L-Arginine to obtain up to 200 mM, preferably from 0.5 to 100 mM,most preferably from 5 to 50 mM;c. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM, mostpreferably from 100 mM to 230 mM; andd. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100 mM,most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/ml, preferably from 5 to 50 mg/ml,most preferably from 10 to 30 mg/ml.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilized product.

In yet another embodiment of the present invention the formulationcomprises

a. N-acetate dissolved in water to obtain final concentrations of fromup to 200 mM, preferably from 0.5 to 100 mM, most preferably from 5 to50 mM L-histidine;b. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM, mostpreferably from 100 mM to 230 mM.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/ml, preferably from 5 to 50 mg/ml,most preferably from 10 to 30 mg/ml.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilized product

The GLP-2 analogues of the invention may also be formulated as a slowrelease implantation device for extended and sustained administration ofthe GLP-2 peptide analogue. Such sustained release formulations may bein the form of a patch positioned externally on the body. Examples ofsustained release formulations include composites of biocompatiblepolymers, such as poly(lactic acid), poly(lactic-co-glycolic acid),methylcellulose, hyaluronic acid, sialic acid, silicate, collagen,liposomes and the like. Sustained release formulations may be ofparticular interest when it is desirable to provide a high localconcentration of a GLP-2 analogue of the invention.

The GLP-2 analogue may be utilized in the form of a sterile-filled vialor ampoule containing an intestinotrophic amount of the peptide, ineither unit dose or multi-dose amounts. The vial or ampoule may containthe GLP-2 analogue and the desired carrier, as an administration readyformulation. Alternatively, the vial or ampoule may contain the GLP-2peptide in a form, such as a lyophilized form, suitable forreconstitution in a suitable carrier, such as sterile water orphosphate-buffered saline.

As an alternative to injectable formulations, the GLP-2 analogue may beformulated for administration by other routes. Oral dosage forms, suchas tablets, capsules and the like, can be formulated in accordance withstandard pharmaceutical practice. According to the present invention,the GLP-2 analogue is administered to treat individuals that wouldbenefit from growth of small bowel tissue.

Nasal dosage forms can be formulated with addition of enhancers, such asChitosan or detergents such as Tween 20, Tween 80, Poloxamers, e.g.,Pluronic F-68, Pluronic F-127; Brij 35, Brij 72, cremophor EL.

The peptide compounds of the present invention may be used alone, or incombination with compounds having an anti-inflammatory effect. Withoutbeing bound by theory, it is envisioned that such combination treatmentmay enforce the beneficial treatment effects of the present peptideanalogues.

The therapeutic dosing and regimen most appropriate for patienttreatment will of course vary with the disease or condition to betreated, and according to the patient's weight and other parameters.Without wishing to be bound by any particular theory, it is expectedthat doses, in the μg/kg range, and shorter or longer duration orfrequency of treatment may produce therapeutically useful results, suchas a statistically significant increase particularly in small bowelmass. In some instances, the therapeutic regimen may include theadministration of maintenance doses appropriate for preventing tissueregression that occurs following cessation of initial treatment. Thedosage sizes and dosing regimen most appropriate for human use may beguided by the results obtained by the present invention, and may beconfirmed in properly designed clinical trials.

An effective dosage and treatment protocol may be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

A human dose of a GLP-2 peptide according to the invention may in oneembodiment be from about 10 μg/kg body weight/day to about 10 mg/kg/day,preferably from about 50 μg/kg/day to about 5 mg/kg/day, and mostpreferably about 100 μg/kg/day to 1 mg/kg/day.

Medical Conditions

The peptides of the present invention are useful as a pharmaceuticalagent for preventing or treating an individual suffering fromgastro-intestinal disorders, including the upper gastrointestinal tractof the esophagus by administering an effective amount of a GLP-2analogue, or a salt thereof as described herein. The stomach andintestinal-related disorders include ulcers of any etiology (e.g.,peptic ulcers, drug-induced ulcers, ulcers related to infections orother pathogens), digestion disorders, malabsorption syndromes,short-bowel syndrome, cul-de-sac syndrome, inflammatory bowel disease,celiac sprue (for example, arising from gluten induced enteropathy orceliac disease), tropical sprue, hypogammaglobulinemic sprue, enteritis,ulcerative colitis, small intestine damage and chemotherapy induceddiarrhea/mucositis (CID).

As mentioned above in general, individuals who would benefit fromincreased small intestinal mass and consequent and/or maintenance ofnormal small intestine mucosal structure and function are candidates fortreatment with the present GLP-2 analogues. Particular conditions thatmay be treated with GLP-2 analogue include the various forms of sprueincluding celiac sprue, which results from a toxic reaction toalpha-gliadin from heat, may be a result of gluten-induced enteropathyor celiac disease, and is marked by a significant loss of villae of thesmall bowel; tropical sprue, which results from infection and is markedby partial flattening of the villae; hypogammaglobulinemic sprue, whichis observed commonly in patients with common variable immunodeficiencyor hypogammaglobulinemia and is marked by significant decrease in villusheight. The therapeutic efficacy of the GLP-2 analogue treatment may bemonitored by enteric biopsy to examine the villus morphology, bybiochemical assessment of nutrient absorption, by patient weight gain,or by amelioration of the symptoms associated with these conditions.

Other conditions that may be treated with the GLP-2 analogues of theinvention, or for which the GLP-2 analogues may be usefulprophylactically, include in addition to the above mentioned radiationenteritis, infectious or post-infectious enteritis, and small intestinaldamage due to cancer-chemotherapeutic or toxic agents.

The GLP-2 analogues may also be used for the treatment of malnutrition,for example cachexia and anorexia.

A particular embodiment the invention is concerned with using thepresent peptides for the prevention and/or treatment of intestinaldamage and dysfunction. Such damage and dysfunction is a well-known sideeffect of cancer-chemotherapy treatment. Chemotherapy administration isfrequently associated with unwanted side effects related to thegastronintestinal system such as mucositis, diarrhea, bacterialtranslocation, malabsorption, abdominal cramping, gastrointestinalbleeding and vomiting. These side effects are clinical consequences ofthe structural and functional damage of the intestinal epithelium andfrequently make it necessary to decrease the dose and frequency ofchemotherapy. Administration of the present GLP-2 peptide agonists mayenhance trophic effect in the intestinal crypts and rapidly provide newcells to replace the damaged intestinal epithelium followingchemotherapy. The ultimate goal achieved by administering the presentpeptides is to reduce the morbidity related to gastrointestinal damageof patients undergoing chemotherapy treatment while creating the mostoptimal chemotherapy regime for the treatment of cancer. Concomitantprophylactic or therapeutic treatment may be provided in accordance withthe present invention to patients undergoing or about to undergoradiation therapy.

The stem cells of the small intestinal mucosa are particularlysusceptible to the cytotoxic effects of chemotherapy due to their rapidrate of proliferation (Keefe et al., Gut 2000; 47: 632-7).Chemotherapy-induced damage to the small intestinal mucosa is clinicallyoften referred to as gastrointestinal mucositis and is characterized byabsorptive and barrier impairments of the small intestine. For example,it has been shown that, the broadly used chemotherapeutic agents, 5-FU,irinotecan, and methothrexate increase apoptosis leading to villusatrophy and crypt hypoplasia in the small intestine of rodents (Keefe etal., Gut 47: 632-7, 2000; Gibson et al., J Gastroenterol Hepatol.September; 18(9):1095-1100, 2003; Tamaki et al., J Int Med Res.31(1):6-16, 2003). Chemotherapeutic agents have been shown to increaseapoptosis in intestinal crypts at 24 hours after administration andsubsequently to decrease villus area, crypt length, mitotic count percrypt, and enterocyte height three days after chemotherapy in humans(Keefe et al., Gut 2000; 47: 632-7). Thus, structural changes within thesmall intestine directly lead to intestinal dysfunction and in somecases diarrhea.

Gastrointestinal mucositis after cancer chemotherapy is an increasingproblem that is essentially untreatable once established, although itgradually remits. Studies conducted with the commonly used cytostaticcancer drugs 5-FU and irinotecan have demonstrated that effectivechemotherapy with these drugs predominantly affects structural integrityand function of the small intestine while the colon is less sensitiveand mainly responds with increased mucus formation (Gibson et al., JGastroenterol Hepatol. September; 18(9):1095-1100, 2003; Tamaki et al.,J Int Med Res. 31(1):6-16, 2003).

The novel GLP-2 analogues of the present invention may be useful in theprevention and/or treatment of gastrointestinal injury and side effectsof chemotherapeutic agents. This potentially important therapeuticapplication may apply to currently used chemotherapeutic agents such asbut not limited to: 5-FU, Altretamine, Bleomycin, Busulfan,Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin,Cladribine, Crisantaspase, Cyclophosphamide, Cytarabine, Dacarbazine,Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin,Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxycarbamide,Idarubicin, Ifosfamide, Irinotecan, Liposomal doxorubicin, Leucovorin,Lomustine, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitomycin,Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Pentostatin,Procarbazine, Raltitrexed, Streptozocin, Tegafur-uracil, Temozolomide,Thiotepa, Tioguanine/Thioguanine, Topotecan, Treosulfan, Vinblastine,Vincristine, Vindesine, Vinorelbine, Bleomycin, Busulfan, Capecitabine,Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine,Crisantaspase, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin,Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Etoposide,Fludarabine, Fluorouracil, Gemcitabine, Hydroxycarbamide, Idarubicin,Ifosfamide, Irinotecan, Liposomal doxorubicin, Leucovorin, Lomustine,Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone,Oxaliplatin, Paclitaxel, Pemetrexed, Pentostatin, Procarbazine,Raltitrexed, Streptozocin, Tegafur-uracil, Temozolomide, Thiotepa,Tioguanine/Thioguanine, Topotecan, Treosulfan, Vinblastine, Vincristine,Vindesine, and Vinorelbine.

It is envisioned that the present peptides may be employed in a methodof treating neo-natals by administering an effective amount of a GLP-2analogue, or a salt thereof. Complications with feeding neonatals due tothe lack of development of the intestine may be overcome by using thepresent peptide agonists.

In another embodiment the invention describes a method of treatingDPP-IV (dipeptidylpeptidase-IV) mediated conditions by administering toa patient in need thereof an effective amount of a GLP-2 analogue, or asalt thereof. Such diseases include conditions in which the DPP-IVenzyme is over expressed.

The pharmaceutical composition may in one embodiment be formulated tocause slow release of said GLP-2 analogue, or a salt or derivativethereof as described above.

It is envisaged that the present peptides may be employed in a method oftreating neo-natals by administering an effective amount of a GLP-2analogue, or a salt thereof. Complications with feeding neonatals due tothe lack of development of the intestine may be overcome by using thepresent peptide agonists.

In another embodiment the invention describes a method of treatingDPP-IV (dipeptidylpeptidase-IV) mediated conditions by administering toa patient in need thereof an effective amount of a GLP-2 analogue, or asalt thereof. Such diseases include conditions in which the DPP-IVenzyme is over expressed.

Selecting a Patient for GLP-2 Analogue Therapy

Native GLP-2 and Gly2-GLP-2 have each been observed to accelerate thegrowth of colonic neoplasms in mice (Thulesen et al., Gut 53:1145-50,2004). Thus, it may be desirable to exclude from GLP-2 analogue therapycertain patients who have increased risk of abnormal tissue growth suchas growth in the gastrointestinal tract (e.g., patients who have or areat increased risk of developing a gastrointestinal neoplasm such ascolon cancer). Accordingly, the invention features methods fordetermining whether a patient is eligible for receiving GLP-2 analoguetherapy. The methods include determining whether a patient has or is atincreased risk of developing a stomach or gastrointestinal disorder anddetermining whether the patient has or is at risk of developing aabnormal tissue growth, where having or being at increased risk ofdeveloping a abnormal tissue growth indicates that the patient may beineligible for receiving the GLP-2 analogue therapy. The invention alsofeatures kit that includes a GLP-2 analogue (e.g., a GLP-2 analoguedescribed herein) and instructions for administering the GLP-2 analogueto a patient not having or not at an increased risk of developingabnormal tissue growth (e.g., caused by a neoplasm such as coloncancer).

Diagnosis of a Stomach or Gastrointestinal Disorder

A stomach or bowel related disorder can be diagnosed by any means knownin the art. Ulcers, for example, may be diagnosed by a barium x-ray ofthe esophagus, stomach, and duodenum, by endoscopy, or by blood, breath,and stomach tissue tests (e.g., to detect the presence of Helicobacterpylon). Malabsorption syndromes can be diagnosed by stool tests or bloodtests which monitor nutrient levels, where increased levels of fat instool or low levels of nutrients in blood are diagnostic of amalabsorption syndrome. Celiac sprue can be diagnosed by antibody testswhich may include testing for antiendomysial antibody (IgA),antitransglutaminase (IgA), antigliadin (IgA and IgG), and total serumIgA. Endoscopy or small bowel bioposy can be used to detect abnormalintestinal lining where symptoms such as flattening of the villi, whichare diagnostic of celiac sprue. Other tests for celiac sprue includeperforming a complete blood count to detect anemia, measuring alkalinephosphatase level, where lowered levels indicate bone loss. Other usefulmarkers for celiac sprue include lowered cholesterol, lowered albumin,increased liver enzymes, and abnormal blood clotting. Tropical sprue canbe diagnosed by detecting malabsorption or infection using small bowelbiopsy. Blood tests showing anemia are likewise indicative of tropicalsprue as are detection of increased fecal fat or decreased levels ofserum calcium, albumin, serum phosphorus, and serum cholesterol.Inflammatory bowel disease can be detected by colonoscopy or by an x-rayfollowing a barium enema, where inflammation, bleeding, or ulcers on thecolon wall are diagnostic of inflammatory bowel diseases such asulcerative colitis or Crohn's disease.

Identification of at-Risk Patient Populations

Once a patient has been identified as a candidate for GLP-2 therapy, itcan be determined if the patient is at increased risk of abnormal tissuegrowth. In particular, the abnormal tissue growth may be in theintestine or may be associated with a neoplasm (e.g., colon cancer).Risk factors for developing, for example, colon cancer include thepresence of colorectal polyps, cancer elsewhere in the body, a previouscolon cancer, a family history of colon cancer, and chronic inflammatorybowel disease (e.g., ulcerative colitis or Crohn's disease), and thepresence of dysplastic cells. Heritable risk factors include familialadenomatous polyposis (FAP), which may be caused by mutations in the APCgene and hereditary nonpolyposis colon cancer (HNPCC), which can becaused by a mutation in one of the hMSH2, hMLH1, hPMSI, and hPMS2 genes.Other factors associated with colon cancer include high-meat, high-fat,or low-fiber diet, inactivity, obesity, smoking, and heavy alcoholintake. Diagnosis of cancer is typically by endoscopy or sigmoidoscopy.

Administration of a GLP-2 Analogue

In some embodiments of the invention, selection of a patient for GLP-2analogue therapy is followed by administration of a GLP-2 analogue(e.g., any GLP-2 analogue described herein) to the patient. Theadministration can be by any route, formaulation, frequency, or amount(e.g., any of those described herein).

EXAMPLES

The following examples are provided to illustrate preferred aspects ofthe invention and are not intended to limit the scope of the invention.

General Peptide Synthesis Apparatus and Synthetic Strategy

Peptides were synthesized batchwise in a polyethylene vessel equippedwith a polypropylene filter for filtration using9-fluorenylmethyloxycarbonyl (Fmoc) as N-α-amino protecting group andsuitable common protection groups for side-chain functionalities.

Solvents

Solvent DMF (N,N-dimethylformamide, Riedel de-Häen, Germany) waspurified by passing through a column packed with a strong cationexchange resin (Lewatit S 100 MB/H strong acid, Bayer AG Leverkusen,Germany) and analyzed for free amines prior to use by addition of3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH) giving rise toa yellow color (Dhbt-O-anion) if free amines are present. Solvent DCM(dichloromethane, analytical grade, Riedel de-Häen, Germany) was useddirectly without purification. Acetonitril (HPLC-grade, Lab-Scan, DublinIreland) was used directly without purification.

Amino Acids

Fmoc-protected amino acids were purchased from Advanced ChemTech (ACT)in suitable side-chain protected forms.

Coupling Reagents

Coupling reagent diisopropylcarbodiimide (DIC) was purchased from Riedelde-Häen, Germany.

Solid Supports

Peptides were synthesized on TentaGel S resins 0.22-0.31 mmol/g.TentaGel S-Ram, TentaGel S RAM-Lys(Boc)Fmoc (Rapp polymere, Germany)were used in cases where a C-terminal amidated peptide was preferred,while TentaGel S PHB, TentaGel S PHB Lys(Boc)Fmoc were used when aC-terminal free carboxylic acid was preferred.

Catalysts and Other Reagents

Diisopropylethylamine (DIEA) was purchased from Aldrich, Germany,piperidine and pyridine from Riedel-de Häen, Frankfurt, Germany.Ethandithiol was purchased from Riedel-de Häen, Frankfurt, Germany.3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH),1-hydroxybenzotriazole (HOBt) (HOAt) were obtained from Fluka,Switzerland. Acetic anhydride was obtained from Fluka.

Coupling Procedures

The amino acids were coupled as in situ generated HObt or HOAt estersmade from appropriate N-a-protected amino acids and HObt or HOAt bymeans of DIC in DMF. Acylations were checked by the ninhydrin testperformed at 80° C. in order to prevent Fmoc deprotection during thetest (Larsen, B. D. and Holm, A., Int. J. Peptide Protein Res. 43, 1994,1-9).

Deprotection of the N-α-Amino Protecting Group (Fmoc).

Deprotection of the Fmoc group was performed by treatment with 20%piperidine in DMF (1×5 and 1×10 min.), followed by wash with DMF (5×15ml, 5 min. each) until no yellow color could be detected after additionof Dhbt-OH to the drained DMF.

Coupling of HOBt-Esters

3 eq. N-α-amino protected amino acid was dissolved in DMF together with3 eq. HObt and 3 eq DIC and then added to the resin.

Cleavage of Peptide from Resin with Acid.

Peptides were cleaved from the resins by treatment with 95%triflouroacetic acid (TFA, Riedel-de Häen, Frankfurt, Germany)-water v/vor with 95% TFA and 5% ethandithiol v/v at r.t. for 2 h. The filteredresins were washed with 95% TFA-water and filtrates and washingsevaporated under reduced pressure. The residue was washed with ether andfreeze dried from acetic acid-water. The crude freeze dried product wasanalyzed by high-performance liquid chromatography (HPLC) and identifiedby mass spectrometry (MS).

Batchwise Peptide Synthesis on TentaGel Resin (PEG-PS).

TentaGel resin (1 g, 0.23-0.24 mmol/g) was placed in a polyethylenevessel equipped with a polypropylene filter for filtration. The resinwas swelled in DMF (15 ml), and treated with 20% piperidine in DMF inorder to remove the initial Fmoc group either on the linker TentaGel SRAM or on the first amino acid on the resin TentaGel S RAM-Lys(Boc)Fmoc.The resin was drained and washed with DMF until no yellow color could bedetected after addition of Dhbt-OH to the drained DMF. The amino acidsaccording to the sequence were coupled as preformed Fmoc-protected HObtesters (3 eq.) as described above. The couplings were continued for 2 h,unless otherwise specified. The resin was drained and washed with DMF(5×15 ml, 5 min each) in order to remove excess reagent. All acylationswere checked by the ninhydrin test as described above. After completedsynthesis the peptide-resin was washed with DMF (3×15 ml, 5 min each),DCM (3×15 ml, 1 min each) and finally diethyl ether (3×15 ml, 1 mineach) and dried in vacuo. The peptide was cleaved from the resin asdescribed earlier and the crude peptide product was analysed andpurified as described below

HPLC Conditions

Gradient HPLC analysis was done using a Hewlett Packard HP 1100 HPLCsystem consisting of an HP 1100 Quaternary Pump, an HP 1100 Autosampler,an HP 1100 Column Thermostat and HP 1100 Multiple Wavelength Detector.Hewlett Packard Chemstation for LC software (rev. A.06.01) was used forinstrument control and data acquisition. The following columns and HPLCbuffer system was used:

Column: VYDAC 238TP5415, C-18, 5 mm, 300 Å 150×4.6 mm. Buffers: A: 0.1%TFA in MQV; B: 0.085% TFA, 10% MQV, 90% MeCN. Gradient: 0-1.5 min. 0% B

-   -   1.5-25 min 50% B    -   25-30 min 100% B    -   30-35 min 100% B    -   35-40 min 0% B        Flow 1, ml/min, oven temperature 40° C., UV detection: I=215 nm.

HPLC Purification of the Crude Peptide

The crude peptide products were purified PerSeptive Biosystems VISIONWorkstation. VISION 3.0 software was used for instrument control anddata acquisition. The following column and HPLC buffer system was used:

Column: Kromasil KR 100 Å, 10 mm C-8, 250×50.8 mm.

Buffer system: Buffers: A: 0.1% TFA in MQV; B: 0.085% TFA, 10% MQV, 90%MeCN.

Gradient: 0-37 min. 0-40% B

Flow 35 ml/min, UV detection: I=215 nm and 280 nm.

Mass Spectroscopy

The peptides were dissolved in super gradient methanol (Labscan, Dublin,Ireland), milli-Q water (Millipore, Bedford, Mass.) and formic acid(Merck, Damstadt, Germany) (50:50:0.1 v/v/v) to give concentrationsbetween 1 and 10 ring/ml. The peptide solutions (20 ml) were analysed inpositive polarity mode by ESI-TOF-MS using a LCT mass spectrometer(Micromass, Manchester, UK) accuracy of +/−0.1 m/z.

General Synthetic Procedure

In all syntheses dry TentaGel-S-Ram resin (1 g, 0.22-0.31 mmol/g) wasplaced in a polyethylene vessel equipped with a polypropylene filter forfiltration. The resin was swelled in DMF (15 ml), and treated with 20%piperidine in DMF to secure the presence of non-protonated amino groupson the resin. The resin was drained and washed with DMF until no yellowcolour could be detected after addition of Dhbt-OH to the drained DMF.The amino acids according to the sequence were coupled as preformedFmoc-protected HOBt esters (3 eq.) as described above. The couplingswere continued for 2 h, unless otherwise specified. The resin wasdrained and washed with DMF (5×15 ml, 5 min each) in order to removeexcess reagent. All acylations were checked by the ninhydrin testperformed at 80° C. After completed synthesis the peptide-resin waswashed with DMF (3×15 ml, 5 min each), DCM (3×15 ml, 1 min each) andfinally diethyl ether (3×15 ml, 1 min each) and dried in vacuo. Thepeptide was then cleaved from the resin as described above andfreeze-dried.

After purification using preparative HPLC as described above, thepeptide product was collected and the identity of the peptide wasconfirmed by ES-MS. This procedure was used for the synthesis of allpeptides exemplified further below.

Compounds Synthesised

Using the above techniques compounds 1809 to 1861 (SEQ ID NOS:1-49) andreference compound 1559 (H-[Gly2]hGLP-2-OH) (SEQ ID NO:54) weresynthesised using the methods described above (Table 1).

TABLE 1 Compounds synthesized Mw Mw purity Compound # Sequence calcfound % Yield* 1559  H-HGDGSFSDEMNTILDNLAARDFINWLIQTKITD-NH2 3749.803749.16 95 5.4 (SEQ ID NO: 54) 1809 H-HGEGTFSDELATILDALAARDFIAWLIATKITDKKKKKK-NH2 4341.42 4341.62 96 46.5(SEQ ID NO: 1) 1810  H-HGEGTFSDELATILDALAARIFIAWLIATKKKKKKK-NH2 4010.324010.63 91 20 (SEQ ID NO: 2) 1811 H-HGDGSPSDELATILDALAARDFIAWLIATKITD-NH2 3494.8 3494.13 94 44.7(SEQ ID NO: 3) 1812  H-HGEGSFSDELATILDNLAARDFIAWLIQTKITD-NH2 3658.863658.3 91 8 (SEQ ID NO: 4) 1813  H-HGDGSFSDELATILDALAARDFIAWLIATKITD-NH23544.82 3545 95 15.9 (SEQ ID NO: 5) 1814 H-HGDGSFSDELATILDNLAARDFIAWLIATKITD-NH2 3587.83 3588 92 31(SEQ ID NO: 6) 1815  H-HGEGSFSDELATILDALAARDFIAWLIATKITD-NH2 3558.843559 95 33 (SEQ ID NO: 7) 1818 H-HGDGSFSSELATILDNLAARDFIAWLIQTKKKKKKK-NH2 4056.26 4056.25 92 68.3(SEQ ID NO: 8) 1819  H-HGDGSFSDELSTILDNLAARDFIAWLIQTKKKKKKK-NH2 4100.254100.13 92 38.7 (SEQ ID NO: 9) 1820 H-HGDGSFTSELATILDNLAARDFIAWLIQTKKKKKKK-NH2 4070.28 4070.38 94 63.3(SEQ ID NO: 10) 1821  H-HGDGSFSDELKTILDNLAARDFIAWLIQTKKKKKKK-NH2 4141.324141.5 93 57.3 (SEQ ID NO: 11) 1822 H-HGDGSFTDELKTILDNLAARDFIAWLIQTKKKKKKK-NH2 4155.33 4155.13 94 72(SEQ ID NO: 12) 1823  H-HGDGSFTSELKTILDNLAARDFIAWLIQTKKKKKKK-NH2 4127.344127.9 95 100.3 (SEQ ID NO: 13) 1824 H-HGDGSFIDELATILDNLAARDFIAWLIQTKKKKKKK-NH2 4098.27 4098.25 95 33.9(SEQ ID NO: 14) 1825  H-HGDGSFSSELATILDNLAARDFIAWLIQTK-NH2 3287.693287.75 92 82.0 (SEQ ID NO: 15) 1826 H-HGDGSFSDELNTILDNLAARDFIAWLIQTKITDKKKKKK-NH2 4456.42 4456.38 93 68.9(SEQ ID NO: 16) 1827  H-HGDGSFTDELSTILDNLAARDFIAWLIQTKKKKKKK-NH2 4114.274114.63 93 20.2 (SEQ ID NO: 17) 1828 H-HGDGSFTSELSTILDNLAARDFIAWLIQTKKKKKKK-NH2 4086.27 4086.63 94 84.5(SEQ ID NO: 18) 1829  H-HGDGSFSSELSTILDNLAARDFIAWLIQTKKKKKKK-NH2 4072.264072.5 95 51.8 (SEQ ID NO: 19) 1830 H-HGDGSFSDELSTILDNLAARDFIAWLIQTK-NH2 3331.68 3331.88 94 26.4(SEQ ID NO: 20) 1831  H-HGDGSFTDELSTILDNLAARDFIAWLIQTK-NH2 3345.73345.38 94 46.0 (SEQ ID NO: 21) 1832 H-HGDGSFTSELSTILDNLAARDFIAWLIQTK-NH2 3317.7 3117.88 94 17.5(SEQ ID NO: 22) 1833  H-HGDGSFSSELSTILDNLAARDFIAWLIQTK-NH2 3303.693304.13 97 34.5 (SEQ ID NO: 23) 1834 H-HGDGSFTSELATILDNLAARDFIAWLIQTK-NH2 3301.71 3301.75 94 16.0(SEQ ID NO: 24) 1835  H-HGDGSFSDELKTILDNLAARDFIAWLIQTK-NH2 3372.753373.13 94 89.7 (SEQ ID NO: 25) 1836 H-HGDGSFTDELKTILDNLAARDFIAWLIQTK-NH2 3386.76 3386.88 92 26.0(SEQ ID NO: 26) 1839  H-HGDGSFSDELATILDNLAARDFIAWLIQTKITDKKKKKK-NH24413.42 4413.88 92 18.2 (SEQ ID NO: 27) 1840 H-HGDGSFSDELATILDNLAARDFIAWLIQTKITD-NH2 3644.85 3644.88 95 21.3(SEQ ID NO: 28) 1841  H-HGDGSFSDELATILDNLAARDFIAWLIQTK-NH2 3315.693315.88 91 73.6 (SEQ ID NO: 29) 1842 H-HGDGSFTSELKTILDNLAARDFIAWLIQTK-NH2 3358.77 3358.88 97 26.3(SEQ ID NO: 30) 1843  H-HGDGSFTDELATILDNLAARDFIAWLIQTK-NH2 3329.73329.88 90 23.6 (SEQ ID NO: 31) 1844 H-HGEGTFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 4329.42 4329.63 90 46.0(SEQ ID NO: 32) 1845  H-HGEGTFSDELSTILDALAARDFIAWLIATKITDKKKKKK-NH24357.42 4357.38 93 52.5 (SEQ ID NO: 33) 1846 H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 4315.41 4315.38 90 28.8(SEQ ID NO: 34) 1847  H-HGEGSFSDELSTILDALAARDFIAWLIATKITDKKKKKK-NH24343.4 4343.5 90 59.4 (SEQ ID NO: 35) 1848 H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 4313.43 4313.63 90 230.0(SEQ ID NO: 36) 1849  H-HGEGSFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH24299.41 4299.5 97 68.0 (SEQ ID NO: 37) 1850 H-HGEGSFSDELKTILDALAARDFIAWLIATKITDKKKKKK-NH2 4384.46 4384.63 93 38.0(SEQ ID NO: 38) 1851  H-HGEGTFSDELKTILDALAARDFIAWLIATKITDKKKKKK-NH24398.48 4398.38 95 90.6 (SEQ ID NO: 39) 1852 H-HGEGTFSSELKTILDALAARDFIAWLIATKITDKKKKKK-NH2 4370.48 4370.63 95 63.2(SEQ ID NO: 40) 1853  H-HGEGTFSSELSTILDALAARDFIAWLIATKITD-NH2 3560.853560.13 97 18.0 (SEQ ID NO: 41) 1854 H-HGEGTFSDELSTILDALAARDFIAWLIATKITD-NH2 3588.85 3589.13 96 12.5(SEQ ID NO: 42) 1855  H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH2 3546.843547 96 27.3 (SEQ ID NO: 43) 1856 H-HGEGSFSDELSTILDALAARDFIAWLIATKITD-NH2 3574.83 3575 96 18.0(SEQ ID NO: 44) 1857  H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH2 3544.863544.88 94 39.3 (SEQ ID NO: 45) 1858 H-HGEGSFSSELATILDALAARDFIAWLIATKITD-NH2 3530.84 3530.88 90 43.3(SEQ ID NO: 46) 1859  H-HGEGSFSDELKTILDALAARDFIAWLIATKITD-NH2 3615.893615.13 90 11.4 (SEQ ID NO: 47) 1860 H-HGEGTFSDELKTILDALAARDFIAWLIATKITD-NH2 3629.91 3629.13 92 13.9(SEQ ID NO: 48) 1861  H-HGEGTFSSELKTILDALAARDFIAWLIATKITD-NH2 3601.913601.13 99 16.2 (SEQ ID NO: 49) *)yield; yield/g resin

Example 1. Synthesis of Compound 1846 (SEQ ID NO:34)

(SEQ ID NO: 34) H-His-Gly-Glu-Gly-Ser-Phe-Ser-Ser-Glu-Leu-Ser-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys- Lys-Lys-Lys-NH₂on TentaGel S RAM-Lys(Boc)Fmoc.

Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand treated as described under “batchwise peptide synthesis on TentaGelresin” until finishing the coupling of the N-terminal Histidine. Allcouplings were continued over night. The acylations were checked asearlier described. After completed synthesis and deprotection of theN-terminal Fmoc group the peptide was cleaved from the resin asdescribed above. After purification using preparative HPLC as earlierdescribed, 28.8 mg peptide product was collected with a purity betterthan 90% and the identity of the peptide was confirmed by MS (found M4315.38, calculated M 4315.41).

Example 2. Synthesis of Compound 1848 (SEQ ID NO:36)

(SEQ ID NO: 36) H-His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu-Leu-Ala-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys- Lys-Lys-Lys-NH₂on TentaGel S RAM-Lys(Boc)Fmoc.

Dry TentaGel S RAM-Lys(Boc)Fmoc (0.24 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand treated as described under “batchwise peptide synthesis on TentaGelresin” until finishing the coupling of the N-terminal Histidine. Allcouplings were continued over night. The acylations were checked asearlier described. After completed synthesis and deprotection of theN-terminal Fmoc group the peptide was cleaved from the resin asdescribed above. After purification using preparative HPLC as earlierdescribed, 230 mg peptide product was collected with a purity betterthan 90% and the identity of the peptide was confirmed by MS (found M4313.63, calculated M 4313.43).

Example 3. Synthesis of Compound 1855 (SEQ ID NO:43)

H-His-Gly-Glu-Gly-Ser-Phe-Ser-Ser-Glu-Leu-Ser-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-NH₂(SEQ ID NO:43) on TentaGel S RAM-Asp(OtBu)Fmoc. Dry TentaGel SRAM-Asp(OtBu)Fmoc (0.2 mmol/g, 1 g) was placed in a polyethylene vesselequipped with a polypropylene filter for filtration and treated asdescribed under “batchwise peptide synthesis on TentaGel resin” untilfinishing the coupling of the N-terminal Histidine. All couplings werecontinued over night. The acylations were checked as earlier described.After completed synthesis and deprotection of the N-terminal Fmoc groupthe peptide was cleaved from the resin as described above. Afterpurification using preparative HPLC as earlier described, 27.3 mgpeptide product was collected with a purity better than 96% and theidentity of the peptide was confirmed by MS (found M 3547, calculated M3546.84).

Example 4. Synthesis of Compound 1857 (SEQ ID NO:45)

H-His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu-Leu-Ala-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-NH₂(SEQ ID NO:45) on TentaGel S RAM-Asp(OtBu)Fmoc. Dry TentaGel SRAM-Asp(OtBu)Fmoc (0.2 mmol/g, 1 g) was placed in a polyethylene vesselequipped with a polypropylene filter for filtration and treated asdescribed under “batchwise peptide synthesis on TentaGel resin” untilfinishing the coupling of the N-terminal Histidine. All couplings werecontinued over night. The acylations were checked as earlier described.After completed synthesis and deprotection of the N-terminal Fmoc groupthe peptide was cleaved from the resin as described above. Afterpurification using preparative HPLC as earlier described, 39.3 mgpeptide product was collected with a purity better than 94% and theidentity of the peptide was confirmed by MS (found M 3544.88, calculatedM 3544.86).

Example 5. Synthesis of Compound 1846 A (SEQ ID NO:34) (Acetate Salt)

Counter ion exchange from trifluoroacetate to acetate of Compound 1846(SEQ ID NO:34).

The purified synthetic peptide product of compound 1846 (SEQ ID NO:34)is isolated as a trifluoroacetate salt, due to the presence oftrifluoroacetic acid (0.1% v/v) in the HPLC buffers used for thepurification of the crude synthetic peptide product.

In order to exchange the counter ion trifluoroacetate with acetate, asolution of the peptide was passed through a column packed with strongbase ion exchange resin on the acetate (Dowex 1×8). 365 mg Compound 1 isdissolved in 40 ml water. The solution is passed through a columncontaining 40 ml strong base ion exchange resin on the acetate (Dowex1×8; capacity 1.33 meq/ml resin). The resin is then washed with 4×30 mlwater and the eluate is collected and lyophilized resulting in 312 mgacetate salt with a purity according to HPLC analysis of 97%.

Example 6. Synthesis of Compound 1848 (SEQ ID NO:36) C (Chloride Salt)

Counter ion exchange from trifluoroacetate (Tfa) to chloride (Cl—) ofCompound 1848 (SEQ ID NO:36). 100 mg Compound 1 was dissolved in 50 ml0.1M hydrochloric acid and the resulting solution was lyophilized. Theremanence was dissolved in 50 ml water and lyophilized again resultingin 80 mg of the chloride salt with a purity according to HPLC of 93%.

Example 7—Chemical Stability Testing

The GLP-2 analogues were dissolved in purified water and subsequentlydiluted in solutions containing HCl, NaOH, H₂O₂ or NH₄HCO₃. Thesolutions were incubated at 40° C. to produce hydrolysis, deamidationand oxidation products. The samples were analyzed by RP-HPLC and thepercentage of remaining intact compound was determined as a measure forthe relative stability. The major degradation products were tentativelyidentified by LC-MS. The peptides used are listed in Table 3.

TABLE 2 GLP-2 analogues tested and compared for chemical stability.Compound Batch Monoisotopic Peptide Purity No. No. MW (g/mol) Content(%) RP-HPLC (%) ZP1559 (SEQ ID NO: 54) 70.30 AB and K 1A 3749.80 90 96ZP1820 (SEQ ID NO: 10) 78.65 1A 4070.28 80 94 ZP1834 (SEQ ID NO: 24)78.90 1A 3301.71 88 94 ZP1846 (SEQ ID NO: 34) 107.07 1A 4315.41 80 90ZP1848 (SEQ ID NO: 36) 91.58 1A 4313.43 80 90 ZP1849 (SEQ ID NO: 37)91.60 1A 4299.41 79 97 ZP1855 (SEQ ID NO: 43) 88.49 1A-2-1A 3546.84 8996 ZP1857 (SEQ ID NO: 45) 108.01 X-1A 3544.86 89 94 ZP1858 (SEQ ID NO:46) 108.05 1A 3530.84 89 90

Compound ZP1559 (SEQ ID NO:54), Gly²-GLP-2, was used as a reference forthe other GLP-2 analogues tested. In the GLP-2 analogues, positionswhere the sequence differs from the reference compound ZP1559 (SEQ IDNO:54) are shown in bold font. The sequences are listed in Table 3.

The compounds are listed as pairs according to their sequences and withand without C-terminal —K₆ extension.

TABLE 3 Sequences of Gly²-GLP-2 and GLP-2 analogues. Compound SequenceZP1559 (SEQ ID NO: 54) HGDGS FSDEM NTILD NLAAR DFINW LIQTK ITD-OH ZP1820(SEQ ID NO: 10) HGDGS FTSEL ATILD NLAAR DFIAW LIQTK K ₆-NH ₂ ZP1834 (SEQID NO: 24) HGDGS FTSEL ATILD NLAAR DFIAW LIQTK —NH ₂ ZP1846 (SEQ ID NO:34) HGEGS FSSEL STILD ALAAR DFIAW LIATK ITD K ₆-NH ₂ ZP1855 (SEQ ID NO:43) HGEGS FSSEL STILD ALAAR DFIAW LIATK ITD-NH ₂ ZP1848 (SEQ ID NO: 36)HGEGT FSSEL ATILD ALAAR DFIAW LIATK ITD K ₆-NH ₂ ZP1857 (SEQ ID NO: 45)HGEGT FSSEL ATILD ALAAR DFIAW LIATK ITD-NH ₂ ZP1849 (SEQ ID NO: 37)HGEGS FSSEL ATILD ALAAR DFIAW LIATK ITD K ₆-NH ₂ ZP1858 (SEQ ID NO: 46)HGEGS FSSEL ATILD ALAAR DFIAW LIATK ITD-NH ₂

Chemicals used in the experiment are listed in Table 4.

TABLE 4 Chemicals and reagents used for the analytical procedures.Substance Quality Supplier Product Acetonitrile HPLC grade Riedel-deHaën34851 (MeCN) Trifluoroacetic 99.9% Pierce 28904 Acid (TFA) Formic Acid(FA) 98-100% Merck 1.00264.1000 Hydrochloric acid “Baker Analyzed” J. T.Baker  7088 HCl Sodium Hydroxide “Baker Analyzed” J. T. Baker  7098 NaOHHydrogen Peroxide 30% w/w Sigma-Aldrich H1009 H₂O₂ NH₄HCO₃ 99.0% AnalaR103025E

The water was first de-mineralized to a resistance of ≧18 MΩcm and thenpassed through a Milli-Q system (Millipore, Bedford, USA). The Milli-Qpurified water (MQW) was finally tapped through a 0.22-μm sterile filter(Millipak 40 Gamma Gold, Millipore).

The GLP-2 analogues tested and the reference Gly²-GLP-2, ZP1559 (SEQ IDNO:54), were dissolved in water and subsequently diluted into solutionscontaining HCl, NaOH, H₂O₂ or NH₄HCO₃. The samples were incubated at 40°C. to generate hydrolysis, deamidation and oxidation products,respectively. The compounds were analysed by RP-HPLC for purity of theoriginal main peak and by LC-MS for confirmation of the identity by massof the main peak and major degradation products.

Preparation of Stress Solutions:

-   0.2 M HCl: 4 ml MQW and 1 ml of 1 M HCl.-   0.02 M NaOH: 4 ml MQW and 1 ml of 0.1 M NaOH.-   0.2 M NH₄HCO₃, pH 8: 0.79 g NH₄HCO₃ was dissolved in 50 ml MQW.-   1% H₂O₂: 5.8 ml MQW and 0.2 ml of 30% H₂O₂.

Sample Solutions:

The GLP-2 analogues were first dissolved in MQW to a concentration of 4mg/ml and then further diluted in the stress solutions at a 1:1 ratio(e.g. 125 μl plus 125 μl). The final concentrations were 2 mg/ml of theGLP-2 analogues for stress conditions with 0.1 M HCl, 0.01M NaOH, 0.1 MNH₄HCO₃ and 0.5% H₂O₂, respectively.

The solutions were incubated at 40° C. in the dark and then diluted inEluent A to a concentration of 0.5 mg/ml (addition of 750 μl) prior toanalyses by RP-HPLC and by LC-MS.

TABLE 5 Conditions for stress test. Solution 0.5% 0.1M HCl 0.01M NaOH0.1M NH₄HCO₃ H₂O₂ Temperature 40 40 40 40 (° C.) Storage (days) 12 3 6 3

RP-HPLC

The RP-HPLC analyses were performed on an Agilent Series 1100 HPLCsystem under control of the ChemStation (Revision A.08.03 [847])software from Agilent Technologies, Inc. The raw data and the results ofthe peak integration were deposited on the ChemStore C/S server by theuse of Agilent Technologies Revision B01.03 software.

TABLE 6 The RP-HPLC method. Method file name P2204071.M Column Vydac218MS52, 5 μm, 300 Å, 2.1 × 250 mm Gradient (time; % B) 0; 5, 2; 5, 7;15, 25; 30, 45; 40, 65; 50, 70; 100, 73; 100, 75; 5, 90; 5 Eluent A0.05% TFA, 0.05% FA in MQW Eluent B 0.05% TFA, 0.05% FA in MeCN FlowRate 0.200 ml/min Injection Volume 20 μl Column Temperature 25° C. AutoSampler Temp. 4° C. UV detection 220 nm

LC-MS

Analytical LC-MS analyses were performed on an Agilent Technologies 1100HPLC instrument consisting of an on-line degasser, quaternary gradientpump, an auto sampler, a column oven, an UV detector. The HPLCinstrument was interfaced with a Micromass LCT (ESI-TOF) massspectrometer under control of Masslynx 3.5 software, from MicroMass, UK.

TABLE 7 The LC-MS method Method file name P22_04_071_003.M Column Vydac218MS52, 5 μm, 300 Å, 2.1 × 250 mm Gradient (time; % B) 0; 5, 2; 5, 7;15, 25; 30, 45; 40, 65; 50, 70; 100, 73; 100, 75; 5, 90; 5 Eluent A0.05% TFA, 0.05% FA in MQW Eluent B 0.05% TFA, 0.05% FA in MeCN FlowRate 0.200 ml/min Injection Volume 30 μl Column Temperature 25° C. AutoSampler Temp. 4° C. UV detection 220 nm

TABLE 8 The MS set-up according to SOP 22-3003. Cone voltage 30 VCapillary voltage 3.1 kV Nitrogen nebuliser gas flow 100 L/hrDesolvation gas flow 500 L/hr Desolvation temperature 250° C. Sourceblock temperature 100° C.

The results are shown in Table 9 as the compound purity measured byRP-HPLC after incubation under stress conditions. This purity is ameasure for the remaining intact compound after incubation in stresssolutions, relative to the purity measured a T=0. These results do nottake into account possible hidden degradation products not observed bythis analytical RP-HPLC method.

Major degradation products in the stress test samples were tentativelyidentified by the LC-MS method. Any isomers to the parent compounds andminor degradation products were not observed by this analytical LC-MSmethod. Tentative identifications are listed in Tables 11 to 15.

The GLP-2 analogues tested are listed as pairs according to theirssequences with and without the C-terminal —K₆ extension.

TABLE 9 Observed purity of test compounds after incubation under stressconditions. Purity Purity Purity HCl 0.1M H₂O₂ 0.5% NH₄HCO₃ 0.1MSequence +/− 12 days 3 days 6 days GLP-2 analogues C-terminal K₆ (%) (%)(%) ZP1559 (SEQ ID NO: 54) Reference 57 <5 66 ZP1820 (SEQ ID NO: 10) K₆45 62 85 ZP1834 (SEQ ID NO: 24) — 38 53 91 ZP1846 (SEQ ID NO: 34) K₆ 7064 82 ZP1855 (SEQ ID NO: 43) — 85 14 97 ZP1848 (SEQ ID NO: 36) K₆ 63 6893 ZP1857 (SEQ ID NO: 45) — 86 59 96 ZP1849 (SEQ ID NO: 37) K₆ 64 78 86ZP1858 (SEQ ID NO: 46) — 88 60 91

The results for the compounds incubated in NaOH are not listed, becauseno difference in the stability could be observed. The degradationproducts and the main peak all have the same mass by LC-MS analysis;these compounds were probably racemized over time. The results in Table9 show that the GLP-2 analogues tested in general are more chemicallystable than the Gly²-GLP-2 reference, ZP1559 (SEQ ID NO:54).

During acid hydrolysis the GLP-2 analogues tested are more stable thanthe Gly²-GLP-2 reference, ZP1559 (SEQ ID NO:54), except for the ZP1820(SEQ ID NO:10) and ZP1834 (SEQ ID NO:24). This is mainly due to theamino acid Asp in position 3. Glu rather than Asp may minimize thecleavage between amino acid 3 and 4 Asp-Gly. The other ZP GLP-2analogues tested do have approximately the same stability and with atendency of slightly higher stability for the compounds withoutC-terminal —K₆, ZP1855 (SEQ ID NO:43), ZP1857 (SEQ ID NO:45), and ZP1858(SEQ ID NO:46). This difference is explained by the amino acid inposition 33 Asp. In the compound without C-terminal —K₆, this amino acidis C-terminal and a cleavage between amino acid 32 and 33 Tyr-Asp occursmore slowly than for a cleavage between amino acid 33 and 34 Asp-Lys.The difference in the stability is due to the Asp and not to theC-terminal —K₆.

Under the conditions of accelerated oxidation (H₂O₂, see also Table 10and 13), the GLP-2 analogues tested are much more stable than theGly²-GLP-2 reference, ZP1559 (SEQ ID NO:54). This is probably due tooxidation of Met in position 10 in ZP1559 (SEQ ID NO:54). An exceptionis ZP1855 (SEQ ID NO:47), which shows an unexplainable low stability.This could be specific for the batch of ZP1855 (SEQ ID NO:47) andfurther studies will be needed to explain this.

The stability under conditions promoting deamidation (NH₄HCO₃), theGLP-2 analogues tested are more stable than the Gly²-GLP-2 reference,ZP1559 (SEQ ID NO:54). This is probably due to several deamidation sitesin ZP1559 (SEQ ID NO:54), Asn in position 11, 16 and 24, which are notpresent in the sequences for the GLP-2 analogues tested.

Tentative Identification of Major Degradation Products by LC-MS

TABLE 10 Major cleavage sites in ZP GLP-2 analogues and the reference ZP1559 (SEQ ID NO: 54).ZP1559 HGD↓GS FSDEM N↓TILD NLAAR DFINW LIQTK  ITD-OH (SEQ ID NO: 54)ZP1820 HGD↓GS FTSEL ATILD NLAAR DFIAW LIQTK  K₆-NH₂ (SEQ ID NO: 10)ZP1834 HGD↓GS FTSEL ATILD NLAAR DFIAW LIQTK- NH₂ (SEQ ID NO: 24)ZP1846 HGEGS FSSEL STILD ALAAR DFIAW LIATK  ITD↓K₆-NH₂ (SEQ ID NO: 34)ZP1855 HGEGS FSSEL STILD ALAAR DFIAW LIATK  IT↓D-NH2 (SEQ ID NO: 43)ZP1848 HGEGT FSSEL ATILD ALAAR DFIAW LIATK  ITD↓K₆-NH₂ (SEQ ID NO: 36)ZP1857 HGEGT FSSEL ATILD ALAAR DFIAW LIATK  IT↓D-NH₂ (SEQ ID NO: 45)ZP1849 HGEGS FSSEL ATILD ALAAR DFIAW LIATK  ITD↓K₆-NH₂ (SEQ ID NO: 37)ZP1858 HGEGS FSSEL ATILD ALAAR DFIAW LIATK  IT↓D-NH₂ (SEQ ID NO: 46)

Solutions Stressed by HCl

TABLE 11 GLP-2 analogues incubated 12 days at 40° C. in 0.1M HCl.Measured Theoretical Difference Major/minor abundance GLP-2 analogues MW(Da) MW (Da) Mass (Da) Possible ID suggestion ZP1559 (SEQ ID NO: 54)3749.88 3749.80 +0.08 Major product 3732.75 Na −17.13 Minor, cyclicdeamidation 3751.88 Na +2.00 Minor, 2 x deamidation ZP1820 (SEQ ID NO:10) 4070.13 4070.28 −0.15 Major product A¹-A³⁶ 4071.25 Na +1.12 Minor,deamidation 3761.25 3761.17 +0.08 Minor, hydrolysis A⁴-A³⁶ 2526.752526.56 +0.19 Minor, hydrolysis A¹⁶-A³⁶ 3762.25 3762.16 +0.09 Minor,hydro. A⁴-A³⁶, dea. ZP1834 (SEQ ID NO: 24) 3302.00 3301.71 +0.29 Majorproduct 3303.00 Na +1.00 Minor, deamidation 3283.88 Na −18.12 Minor,cyclic deamidation 3302.88 Na +0.88 Minor, deamidation 2992.88 2992.60−309.12 Major, hydro. A⁴-A³⁰, dea. 2993.88 2993.59 −308.12 Minor,hydrolysis A⁴-A³⁰ 2993.75 2993.59 −308.25 Minor, hydrolysis A⁴-A³⁰ZP1846 (SEQ ID NO: 34) 4315.00 4315.41 +0.59 Major product A¹-A³⁹3547.50 3547.82 −0.32 Minor, hydrolysis A¹-A³³ 3934.88 3935.26 −0.38Minor, hydrolysis A⁵-A³³ 2755.38 2755.70 −0.32 Minor, hydrolysis A¹⁶-A³⁹2158.25 2158.37 −0.12 Minor, hydrolysis A²²-A³⁹ 3155.16 Na −1160.25 Nosuggestion ZP1855 (SEQ ID NO: 43) 3548.13 3546.84 +1.29 Majorproduct/deamida. 3548.13 Na +1.29 Minor, deamidation 3225.13 3223.71+1.42 Minor, hydro. A⁴-A³³, dea. 3433.13 3432.79 +0.34 Minor, hydrolysisA¹-A³² 3354.50 3352.76 +1.76 Minor, hydro. A³-A³³, dea. ZP1857 (SEQ IDNO: 45) 3544.50 3544.86 −0.36 Major product A¹-A³³ 3430.63 3430.81 −0.18Minor, hydrolysis A¹-A³² 3351.38 3350.78 +0.60 Minor, hydrolysis A³-A³³3165.50 3164.71 +0.79 Minor, hydrolysis A⁵-A³³ 3222.50 3221.73 +0.77Minor, hydrolysis A⁴-A³³ 2830.25 2829.56 +0.69 Minor, hydrolysis A⁸-A³³3545.13 Na +0.63 Minor, recemization ZP1849 (SEQ ID NO: 37) 4299.634299.41 +0.22 Major product, A¹-A³⁹ 2755.88 2755.70 +0.18 Minor,hydrolysis, A¹⁶-A³⁸ 3532.00 3531.82 +0.18 Minor, hydrolysis, A¹-A³³2158.38 2159.05 −0.67 Minor, hydrolysis, 2158.37 +0.01 A¹-A²¹ or A²²-A³⁹3976.88 3976.29 +0.59 Minor, hydrolysis, A⁴-A³⁹ 4105.13 4105.33 −0.20Minor, hydrolysis, A³-A³⁹ 3920.50 3919.27 +1.23 Minor, hydro., A⁵-A³⁹,dea. 1561.84 1561.73 +0.11 Minor, hydrolysis, A¹-A¹⁵ ZP1858 (SEQ ID NO:46) 3532.13 3530.84 +1.29 Major product/deamida. na = not available, notheoretical MW was calculated or suggested. * Mass difference is themeasured MW from the theoretical MW for the main peak or for thesuggested compound.

The results in Table 12 show that the measured molecular weight for theGLP-2 analogues tested and the Gly²-GLP-2 reference, ZP1559 (SEQ IDNO:54), correspond to the theoretical molecular weight.

The most abundant degradation products are the cleavage between aminoacids 3 and 4; Asp and Gly in ZP1559 (SEQ ID NO:54), ZP1820 (SEQ IDNO:10), and ZP1834 (SEQ ID NO:24). For the compounds ZP1846 (SEQ IDNO:34), ZP1848 (SEQ ID NO:36), and ZP1849 (SEQ ID NO:37), which are withC-terminal —K₆, degradation products corresponding to a loss ofC-terminal —K₆ (Lys₆) in position 33-39 were detected. For the compoundsZP1855 (SEQ ID NO:43) and ZP1857 (SEQ ID NO:45), which are withoutC-terminal —K₆, degradation products corresponding to a loss ofC-terminal Asp in position 33 were detected.

Minor degradation products detected were cleavage between amino acids 15and 16 (Asp and Asn or Asp and Ala), amino acids 4 and 5 (Gly and Ser),amino acids 21 and 22 (Asp and Phe).

Solutions Stressed by H₂O₂

TABLE 12 GLP-2 analogues incubated 3 days at 40° C. in 0.5% H₂O₂.Measured Theoretical Difference* Major/minor abundance GLP-2 analoguesMW (Da) MW (Da) Mass (Da) Possible ID suggestion ZP1559 (SEQ ID NO: 54)n.d. 3749.80 — No product intact 3766.00 Na +16.2 Major, oxidation of MZP1820 (SEQ ID NO: 10) 4070.63 4070.28 −0.35 Major product 4052.38 Na−18.25 Major, deamidation precursor 4086.50 Na +15.87 Minor, oxidationof W 4068.75 Na −1.88 Minor, 2 x deamidation ZP1834 (SEQ ID NO: 24)3301.75 3301.71 +0.04 Major product 3283.75 Na −18.00 Major, deamidationprecursor 3317.88 Na +16.13 Minor, oxidation of W 3299.75 Na −2.00Minor, 2 x deamidation ZP1846 (SEQ ID NO: 34) 4315.50 4315.41 +0.09Major product 4331.88 Na +16.38 Minor, oxidation of W ZP1855 (SEQ ID NO:43) 3547.00 3546.84 +0.16 Major product 3481.88 Na −65.12 Minor, nosuggest 3410.00 Na −137.00 Minor, A²-A³³ 3563.00 Na +16.00 Minor,oxidation of W ZP1848 (SEQ ID NO: 36) 4313.50 4313.43 +0.07 Majorproduct 4329.63 Na +16.13 Minor, oxidation of W ZP1857 (SEQ ID NO: 45)3545.00 3544.86 +0.14 Major product 3408.00 Na −137.00 Minor, A²-A³³3561.00 Na +16.00 Minor, oxidation of W 3479.88 Na −65.12 Minor, nosuggest ZP1849 (SEQ ID NO: 37) 4299.50 4299.41 +0.09 Major product4315.75 Na +16.25 Minor, oxidation of W ZP1858 (SEQ ID NO: 46) 3531.003530.84 +0.16 Major product 3394.13 Na −136.87 Minor, A²-A³³ 3547.00 Na+16.00 Minor, oxidation of W 3466.00 Na −65.00 Minor, no suggest na =not available, no theoretical MW was calculated or suggested. *Massdifference is the measured MW from the theoretical MW for the main peak.

The results in Table 13 show that the measured molecular weight for theGLP-2 analogues tested and the Gly²-GLP-2 reference, ZP1559 (SEQ IDNO:54), correspond to the theoretical molecular weight. For ZP1559 (SEQID NO:54), a major degradation product with a MW of +16 Da is observed,which could probably be the oxidized products of Met in position 10. ForZP1820 (SEQ ID NO:10) and ZP1834 (SEQ ID NO:24), a major degradationproduct with a MW of +18 Da is observed. They could probably be loss ofwater in the precursor to a deamidation. In addition, minor productswith MW of −2 Da is observed and could be deamidation in two sites.Minor products with MW of +16 Da are observed in all compounds and couldprobably be oxidation of Trp. For the compounds ZP1855 (SEQ ID NO:43),ZP1857 (SEQ ID NO:45), and ZP1858 (SEQ ID NO:46), which all are withoutC-terminal —K₆, minor degradation products with MW of −137 Da and of −65Da are detected, corresponding to a loss of His in position 1 and anunidentified degradation product, respectively.

Solutions Stressed by NaOH

TABLE 13 GLP-2 analogues incubated 3 days at 40° C. in 0.01M NaOH.Measured MW Theoretical Difference* GLP-2 analogues (Da) MW (Da) Mass(Da) Comments ZP1559 (SEQ ID NO: 54) 3750.38 3749.80 +0.58 No change inMW ZP1820 (SEQ ID NO: 10) 4070.63 4070.28 +0.35 No change in MW ZP1834(SEQ ID NO: 24) 3302.00 3301.71 +0.29 No change in MW ZP1846 (SEQ ID NO:34) 4315.63 4315.41 +0.22 No change in MW ZP1855 (SEQ ID NO: 43) 3547.253546.84 +0.41 No change in MW ZP1848 (SEQ ID NO: 36) 4313.88 4313.43+0.45 No change in MW ZP1857 (SEQ ID NO: 45) 3545.13 3544.86 +0.27 Nochange in MW ZP1849 (SEQ ID NO: 37) 4299.88 4299.41 +0.47 No change inMW ZP1858 (SEQ ID NO: 46) 3531.13 3530.84 +0.29 No change in MW na = notavailable, no theoretical MW was calculated or suggested. *Massdifference is the measured MW from the theoretical MW for the main peak.

The LC-MS analyses show the same molecular weight in all peaks for eachcompound. This means that the most abundant degradation products areprobably racemization from L- to D-form of one or more of the aminoacids in the sequence. The main peak can then hide one or severalracemized products, thus no residual purity of intact peptide could bedetermined. No major degradation products from cleavage have beendetected.

Solutions Stressed by NH₄HCO₃

TABLE 14 GLP-2 analogues incubated 6 days at 40° C. in 0.1M NH₄HCO₃, pH8 Measured Theoretical Difference* Major/minor abundance GLP-2 analoguesMW (Da) MW (Da) Mass (Da) Possible ID suggestion ZP1559 (SEQ ID NO: 54)3750.00 3749.80 +0.20 Major product 3751.13 Na +1.13 Minor, possibledeamidation 3751.13 Na +1.13 Minor, possible deamidation ZP1820 (SEQ IDNO: 10) 4070.13 4070.28 −0.15 Major product 4070.13 Na −0.15 Minor,possible racemization ZP1834 (SEQ ID NO: 24) 3302.00 3301.71 +0.29 Majorproduct 3302.00 Na +0.29 Minor, possible racemization ZP1846 (SEQ ID NO:34) 4315.25 4315.41 −0.16 Major product 4315.63 Na +0.38 Minor, possibleracemization ZP1855 (SEQ ID NO: 43) 3547.13 3546.84 +0.29 Major productZP1848 (SEQ ID NO: 36) 4313.75 4313.43 +0.32 Major product ZP1857 (SEQID NO: 45) 3544.75 3544.86 −0.11 Major product ZP1849 (SEQ ID NO: 37)4299.50 4299.41 +0.09 Major product ZP1858 (SEQ ID NO: 46) 3531.003530.84 +0.16 Major product na = not available, no theoretical MW wascalculated or suggested. *Mass difference is the measured MW from thetheoretical MW for the main peak.

The results in Table 14 show that the measured molecular weight for theGLP-2 analogues tested and the Gly²-GLP-2 reference, ZP1559 (SEQ IDNO:54), correspond to the theoretical MW. For ZP1559, minor degradationproducts with a MW of +1 Da are observed, which could probably bedeamidated products. For ZP1820 (SEQ ID NO:10), ZP1834 (SEQ ID NO:24),and ZP1846 (SEQ ID NO:34), minor degradation products with the same MWas for the main compound are observed. They could probably be racemizedproducts or deamidation. However, the MS resolution of the presentinstrument was not adequate to confirm or reject this. In addition,these products could be present in the other compounds, but not detectedas they could be hidden under the main peak.

All the present GLP-2 analogues tested have better chemical stabilitycompared with the reference compound Gly²-GLP-2; 1559 (SEQ ID NO:54),under stress conditions for hydrolysis, oxidation and deamidation. Thecompounds 1820 (SEQ ID NO:10) and 1834 (SEQ ID NO:24) are less stablethan the remaining six candidates due to acid hydrolysis. Highestchemical stability was seen when amino acid A³ was Glu rather than Asp.

No significant difference in the chemical stability of the remaining sixcandidates were observed, except a slightly better stability without—K₆, which were mainly due to a labile site after Asp and the loss of—K₆ in candidates having the C-terminal —K₆.

Example 8. Screening for Intestinal Growth Effects of Compounds in C57BLMice

The ability of the present compounds to stimulate intestinal growth wasdetermined in male C57BL mice. Individual groups (n=6) of mice weregiven 30 nmol/kg of each compound, s.c, twice daily for ten consecutivedays. For comparison purposes other groups of animals were given eitheran equimolar dose of [Gly2]GLP-2 (SEQ ID NO:54) or vehicle (phosphatebuffered saline, pH 7.4) in the same dosing regimen. Twenty-four hoursafter the last dose of compound had been given the mice were sacrificedand the small intestine (from the pylorus to the cecum) and the colon(intestine distal to cecum) was emptied and weighed. To correct forslight difference in body weight (BW), the organ mass of the smallintestine (SI) and colon were expressed relative to BW. Thenon-selective reference compound [Gly2]GLP-2 (SEQ ID NO:54) has beenreported to stimulate gastrointestinal growth in both esophagus,stomach, small intestine and colon and to evaluate differences in growthpattern induced by compounds, the small intestine-colon sensitivityindex of compound X was calculated as:

(SI/Colon)_(X)/(SI/Colon)_([Gly2]GLP-2)%

Compounds with a small intestine-colon sensitivity greater than or equalto 1.05 were considered relatively selective for the small intestine(Table 15).

Compounds with a small intestine-colon sensitivity smaller than or equalto 0.95 were considered relatively selective for the colon (Table 15).

TABLE 15 List of selective GLP-2 analogue compounds. Position 1 2 3 4 56 7 8 9 10 11 12 13 14 15 16 17 18 19 Non-selective reference compound =[Gly2]GLP-2 H G D G S F S D E M N T I L D N L A ASmall intestine selective compounds 1844 H G E G T F S S E L S T I L D AL A A 1845 H G E G T F S D E L S T I L D A L A A 1846 H G E G S F S S EL S T I L D A L A A 1848 H G E G T F S S E L A T I L D A L A A 1849 H GE G S F S S E L A T I L D A L A A 1850 H G E G S F S D E L K T I L D A LA A 1851 H G E G T F S D E L K T I L D A L A A 1852 H G E G T F S S E LK T I L D A L A A 1853 H G E G T F S S E L S T I L D A L A A 1855 H G EG S F S S E L S T I L D A L A A 1857 H G E G T F S S E L A T I L D A L AA 1858 H G E G S F S S E L A T I L D A L A A 1859 H G E G S F S D E L KT I L D A L A A Colon selective compounds 1830 H G D G S F S D E L S T IL D N L A A 1831 H G D G S F T D E L S T I L D N L A A 1835 H G D G S FS D E L K T I L D N L A A 1836 H G D G S F T D E L K T I L D N L A A1839 H G D G S F S D E L A T I L D N L A A 1840 H G D G S F S D E L A TI L D N L A A 1841 H G D G S F S D E L A T I L D N L A A 1843 H G D G SF T D E L A T I L D N L A A Position 20 21 22 23 24 25 26 27 28 29 30 3132 33   %[Gly2]GLP-2 Non-selective reference compound = [Gly2]GLP-2 R DF I N W L I Q T K I T D OH 100 100 100Small intestine selective compounds 1844 R D F I A W L I A T K I T D K6NH2 106  89 119 1845 R D F I A W L I A T K I T D K6 NH2 105  98 107 1846R D F I A W L I A T K I T D K6 NH2 120 102 118 1848 R D F I A W L I A TK I T D K6 NH2 109  93 117 1849 R D F I A W L I A T K I T D K6 NH2 109 95 115 1850 R D F I A W L I A T K I T D K6 NH2 103  89 116 1851 R D F IA W L I A T K I T D K6 NH2 109  96 114 1852 R D F I A W L I A T K I T DK6 NH2 106  97 109 1853 R D F I A W L I A T K I T D NH2 101  96 105 1855R D F I A W L I A T K I T D NH2 106  88 120 1857 R D F I A W L I A T K IT D NH2 117  90 130 1858 R D F I A W L I A T K I T D NH2 104  96 1081859 R D F I A W L I A T K I T D NH2  83  73 114Colon selective compounds 1830 R D F I A W L I Q T K NH2 100 106  941831 R D F I A W L I Q T K NH2  90 102  88 1835 R D F I A W L I Q T KNH2  96 105  91 1836 R D F I A W L I Q T K NH2  94 105  90 1839 R D F IA W L I Q T K I T D K6 NH2 112 133  84 1840 R D F I A W L I Q T K I T DNH2 113 131  86 1841 R D F I A W L I Q T K NH2 113 126  90 1843 R D F IA W L I Q T K NH2 111 130  85

Results

The intestinal growth effects of the present compounds according to theinvention were determined based on the ability of the peptides todose-dependently increase SI mass relative to the effect of equimolardoses of the non-selective reference compound [Gly₂]GLP-2 (SEQ IDNO:54).

The findings from this study demonstrate that GLP-2 variants havingamino acid substitutions at positions 8, 16, 24 and/or 28 of thewild-type GLP-2 sequence have increased biological activity compared to[Gly2]GLP-2 (SEQ ID NO:54) in C57BL mice.

Example 9. Dose-Response Effect of Selected Compounds on IntestinalGrowth in C57BL Mice

1820 (SEQ ID NO:10), 1855 (SEQ ID NO:43), 1846 (SEQ ID NO:34), 1858 (SEQID NO:46), 1849 (SEQ ID NO:37), 1848 (SEQ ID NO:36), and 1857 (SEQ IDNO:45) were selected as lead compounds since these compounds bothincreased small intestine mass relative to [Gly2]GLP-2 (SEQ ID NO:54)(Example 8) and had increased chemical stability relative to [Gly2]GLP-2(SEQ ID NO:54) under stressful conditions (Example 7). The dose-responseeffect of 1820 (SEQ ID NO:10), 1855 (SEQ ID NO:43), ZP1846 (SEQ IDNO:34), 1858 (SEQ ID NO:46), 1849 (SEQ ID NO:37), 1848 (SEQ ID NO:36),and 1857 (SEQ ID NO:45) on small intestinal mass was determined in maleC57BL mice. Individual groups (n=6) of mice were given 5, 15, 45, 135 or405 nmol/kg of each compound, s.c, twice daily for three consecutivedays. For comparison purposes other groups of animals were given eitherequimolar doses of [Gly2]GLP-2 (SEQ ID NO:54) or vehicle (phosphatebuffered saline, pH 7.4) in the same dosing regimen. Twenty-four hoursafter the last dose of compound had been given the mice were sacrificedand the small intestine (from the pylorus to the cecum emptied andweighed to determine the effect on small intestinal mass.

Results

The effect of 1820 (SEQ ID NO:10), 1855 (SEQ ID NO:43), 1846 (SEQ IDNO:34), 1858 (SEQ ID NO:46), 1849 (SEQ ID NO:37), 1848 (SEQ ID NO:36),and 1857 (SEQ ID NO:45) on small intestine mass, relative to the effectof the reference compound, [Gly2]GLP-2 (SEQ ID NO:54) is shown in FIGS.1 to 5. At each of the doses tested the effect of [Gly₂]GLP-2 (SEQ IDNO:54) on small intestine mass is standardised at 100%. The intestinalgrowth effects of the compounds 1820 (SEQ ID NO:10), 1855 (SEQ IDNO:43), 1846 (SEQ ID NO:34), 1858 (SEQ ID NO:46), 1849 (SEQ ID NO:37),1848 (SEQ ID NO:36), and 1857 (SEQ ID NO:45) according to the inventionwere determined based on the ability of the peptides to dose-dependentlyincrease SI mass relative to the effect of equimolar doses of[Gly₂]GLP-2 (SEQ ID NO:54). Based on these findings we can conclude thatGLP-2 analogues containing 8 substitutions (G2, E3, T5, L10, A11, A16,A24, A28 with regard to GLP-2 1809 (SEQ ID NO:1)) give rise to asignificant increase in the weight of the small intestine compared tomice treated with [Gly2]GLP-2 (SEQ ID NO:54).

In particular, substitution of Asp3 for Glu and Asn16 for Ala and Gln28for Ala effect the increase in the weight of the small intestine in aselective manner compared to the colon (1839 (SEQ ID NO:27) or 1840 (SEQID NO:28) in comparison with 1809 (SEQ ID NO:1)). Thus, substitution ofthe three amino acids Asp3, Asn16 and Gln28 results in a selectiveincrease of the small intestine weight relative to the colon mass.

Furthermore, substitution of the Asp8 for Serine results in anadditional increase in the selectivity, thus resulting in asupplementary increase in the weight of the small intestine withoutsignificant effecting the weight of the colon (1818 (SEQ ID NO:8), 1820(SEQ ID NO:10), 1844 (SEQ ID NO:32), 1846 (SEQ ID NO:34), 1848 (SEQ IDNO:36), 1849 (SEQ ID NO:37), 1852 (SEQ ID NO:40), 1853 (SEQ ID NO:41),1855 (SEQ ID NO:43), 1858 (SEQ ID NO:46)).

FIGS. 1 to 4 show the results of experiments in which the dose responseeffect of exemplified compounds 1846 (SEQ ID NO:34), 1855 (SEQ IDNO:43), 1848 (SEQ ID NO:36), 1857 (SEQ ID NO:45), 1849 (SEQ ID NO:37) onthe SI-BW to colon-BW ratio is shown relative to reference compound[Gly₂]GLP-2 (SEQ ID NO:54). At each of the doses tested the effect of[Gly₂]GLP-2 (SEQ ID NO:54) on small intestine mass is standardized at100%. All of the exemplified compounds share modifications at positions8, 16 and/or 28 and show increased selectivity for causing growth of thesmall intestine relative to the colon.

Example 10—Dose Response Effect of 1846 (SEQ ID NO:34), 1848 (SEQ IDNO:36), 1855 (SEQ ID NO:43), and 1857 (SEQ ID NO:45) on Small IntestinalAtrophy in Mice Administered 5-FU

The rapid rate of proliferation of the small intestinal stem cells,makes them a susceptible target to the cytotoxic effects of thechemotherapeutic agents used in anti-cancer therapies. Consequently,clinical use of the chemotherapeutic agent 5-fluorouracil (5-FU) isfrequently associated with small intestinal injury (atrophy and diarrheain cancer patients. We have previously shown that i.p. administration of50 mg/kg 5-FU once daily for four days induces significant smallintestinal atrophy in C57BL mice. The effect of the lead compounds, 1846(SEQ ID NO:34), 1848 (SEQ ID NO:36), 1855 (SEQ ID NO:43), and 1857 (SEQID NO:45) on 5-FU-induced small intestinal atrophy was investigated inmice. We have previously shown that i.p. administration of 50 mg/kg 5-FUonce daily for four days induces significant small intestinal atrophy inC57BL mice. 1846 (SEQ ID NO:34), 1848 (SEQ ID NO:36), 1855 (SEQ IDNO:43), or 1857 (SEQ ID NO:45) were administered twice daily for threedays prior to 5-FU and for four days together with 5-FU administration.The lead compounds were each administered at five different doses (5,15, 45, 135 and 405 nmol/kg) that have been previously shown toeffectively stimulate small intestine growth in healthy mice (Example9). For comparison purposes a group of animals were treated with 405nmol/kg [Gly2]GLP-2 (SEQ ID NO:54). To determine the effect of 5-FU onthe small intestine a group of animals were given 5-FU I alone and leftuntreated (5-FU controls) and another group of animals were only givenvehicle (PBS controls).

Results

5-FU induced a significant decrease in SI-BW and SI length in C57BLmice, relative to PBS controls. The dose-response effect of 1846 (SEQ IDNO:34), 1848 (SEQ ID NO:36), 1855 (SEQ ID NO:43), or 1857 (SEQ ID NO:45)on SI-BW and SI length in mice administered 5-FU is shown in FIGS. 6 to9. The effect of 405 nmol/kg [Gly2]GLP-2 (SEQ ID NO:54) is also shown.1846 (SEQ ID NO:34), 1848 (SEQ ID NO:36), 1855 (SEQ ID NO:43), or 1857(SEQ ID NO:45) dose-dependently prevented 5-FU-induced SI atrophy andmaintained SI-BW at levels similar to PBS controls. ZP1848 (SEQ IDNO:36), ZP1855 (SEQ ID NO:43), and ZP1857 (SEQ ID NO:45), given at anequimolar dose, were significantly more efficacious than 405 nmol/kg[Gly2]GLP-2 (SEQ ID NO:54) on SI-BW. 1848 (SEQ ID NO:36) and 1857 (SEQID NO:45), were significantly more efficacious than 405 nmol/kg[Gly2]GLP-2 (SEQ ID NO:54) on SI-length.

Example 11: Dose Response Effect of 1846 (SEQ ID NO:34) on SmallIntestinal Atrophy and Diarrhea in SD Rats Administered 5-FU

The effect of the clinical candidate, 1846 (SEQ ID NO:34), on5-FU-induced small intestinal atrophy and diarrhea was investigated inSD rats. We have previously shown that i.p. administration of 75 mg/kg5-FU once daily for four days induces significant small intestinalatrophy and diarrhea in SD rats. 1846 (SEQ ID NO:34) (16, 80 and 400nmol/kg/d; n=20 rats/dose group) was administered twice daily for threedays prior to 5-FU and for four days together with 5-FU administration.5-FU controls and PBS controls were included in the study. Twenty-fourhours after the last dose of 1846 (SEQ ID NO:34) was given a subset ofanimals were sacrificed to determine the effect of 1846 (SEQ ID NO:34)on small intestinal atrophy. To determine the effect of 1846 (SEQ IDNO:34) on diarrhea all the animals were observed twice daily (morningand evening), during the dosing period and for an additional six days.At each observation period each animal was given a score (0, nodiarrhea, 1 (mild), fecal staining around the anus, 2 (moderate), fecalstaining on the hind limbs and tail and 3 (severe) fecal staining on thefront limbs and abdomen) that indicated whether or not the animal haddiarrhea and the severity of the diarrhea

Results

5-FU induced a significant decrease in SI-BW and SI length and induceddiarrhea, in SD rats, relative to PBS controls. The dose-response effectof 1846 (SEQ ID NO:34) on 5-FU induced small intestinal atrophy anddiarrhea is shown in FIGS. 10 and 11. 1846 (SEQ ID NO:34)dose-dependently prevented 5-FU induced small intestinal atrophy andmaintained SI-BW and SI length at levels similar to the controls. At thehighest dose (400 nmol/kg) administered, 1846 (SEQ ID NO:34), decreasedthe incidence and severity of diarrhea in rats administered 5-FU.

Example 12. Dose Response Effect of 1846 (SEQ ID NO:34) on Crypt-VillusLength and Muscularis Thickness in the Small Intestine of SD Rats

The effect of the clinical candidate, 1846 (SEQ ID NO:34), oncrypt-villus length and muscularis thickness in the small intestine ofSD rats was investigated. 1846 (SEQ ID NO:34) (0.62, 3.41 or 6.2mg/kg/day, n=6 rats/dose group) was administered as an i.v. bolus, oncedaily for five consecutive days. Twenty-four hours after the last dosehad been administered the rats were sacrificed and a 1 cm biopsy wasexcised from the jejunum (30 cm distal from the gastric duodenaljunction) and from the ileum (30 cm proximal from the ileocaecaljunction) for histological processing.

Results

The dose-response effect of 1846 (SEQ ID NO:34) on crypt-villus lengthand muscularis thickness in the jejunum and ileum is shown in FIG. 12.1846 (SEQ ID NO:34) dose-dependently increased mean crypt-villus lengthin the jejunum and ileum muscularis thickness in the ileum.

Example 13. Effect of 1848 (SEQ ID NO:36) on Markers of Small IntestinalInflammation in an Indomethacin-Induced Model of Crohn's Disease

Crohn's disease is a chronic disease that causes episodic inflammationof the small intestine. The effect of the GLP-2 analogue 1848 (SEQ IDNO:36) on small intestinal inflammation in an indomethacin-induced modelof Crohn's disease was investigated. We have previously shown thatadministration of indomethacin (s.c, once daily for 2 days) inducessmall intestinal inflammation characterized by ulcerations and,increases in the pro-inflammatory cytokine, tumour necrosis factor alpha(TNF-α).

To determine the effect of ZP1848 (SEQ ID NO:36) on ulceration 1848 (SEQID NO:36) (8, 40 and 200 nmol/kg, s.c, twice daily (9:00 and 16:00)) wasgiven for 4 days prior to the first dose of indomethacin and for anadditional two days together with indomethacin. The corticosteroid,prednisolone (10 mg/kg, p.o) was used as a positive control sincecorticosteroids are commonly used in in the treatment of activeinflammation in Crohn's disease. In addition a group of animals weregiven both 1848 (SEQ ID NO:36) (200 nmol/kg) and prednisolone todetermine the effects of a combination treatment. Twenty four hoursafter the last dose of 1848 (SEQ ID NO:36) had been given the animalswere sacrificed the small intestine was gently flushed clean and fixed.To determine the extent of ulceration the intestine was cut open alongthe antimesenteric margin, suspended on a polypropylene plate andsurface-stained with Alcian Green 3BX. Starting at the pylorus, thesmall intestine was scanned and the shape (circular vs. linear) and size(circular ulcers: diameter, linear ulcers: length×width) of all ulcerswas measured using a standard ruler (resolution: 0.5 mm). An ulcer wasdefined as an area, which lacked epithelial surface. Finally the totaldamaged area was calculated for each animal by summation of the areas ofall individual ulcers.

To determine the effect of 1848 (SEQ ID NO:36) on the concentrations ofTNF-α in the small intestine 1848 (SEQ ID NO:36) and Indomethacin weregiven as described above. At sacrifice, however the small intestine wasseparated into three segments of equal length (proximal, mid anddistal). TNF-α concentrations were measured in each of the individualsegments using a commercially available ELISA kit.

Results

Indomethacin caused a strong induction of small intestinal ulcers,compared to the control group ((estimated extent of ulceration 333±21mm² vs. 10 mm². The effect of 1846 (SEQ ID NO:34) on the estimatedextent of ulceration (mm²) is shown in FIG. 13. Treatment with 1848 (SEQID NO:36) (8 nmol/kg, 40 nmol/kg and 200 nmol/kg, significantlydecreased the extent of ulceration (230±12 mm², 216±17 mm², and 178±17mm² respectively, p<0.001 vs. indomethacin). At the highest dose (200nmol/kg) used ZP1848 (SEQ ID NO:36) was more effective than the positivecontrol, prednisolone (p<0.05).

Indomethacin caused an approx. 2.9-fold increase in tissue levels ofTNF-α in the proximal segment (97±14 pg/mg protein) compared to controlanimals (34±7 pg/mg protein, p<0.05 vs. indomethacin). The effect of1846 (SEQ ID NO:34) on small intestinal TNF-α concentrations is shown inFIG. 14. Treatment with 1848 (SEQ ID NO:36) (8, 40 or 200 nmol/kg),significantly reduced tissue levels of TNF-α (45±14 pg/mg protein, 44±9pg/mg protein and 45±7 pg/mg protein, respectively) with no significantdifference between the 3 doses.

Indomethacin caused an approx. 3.2-fold increase in tissue levels ofTNF-α in the mid segment (108±9 pg/mg protein) compared to controlanimals (34±6 pg/mg protein, p<0.05 vs. indomethacin). 1848 (SEQ IDNO:36) (40 or 200 nmol/kg) significantly reduced tissue levels of TNF-αrespectively, p<0.05 vs. indomethacin)

Indomethacin caused an approx. 1.7-fold increase in tissue levels ofTNF-α in the distal segment (75±5 pg/mg protein) compared to controlanimals (45±3 pg/mg protein, p<0.05 vs. indomethacin). 1848 (SEQ IDNO:36) had an inhibitory effect on TNF-α levels in the distal segmentbut the effect was less pronounced compared to the other segments.Prednisolone alone did not significantly affect tissue levels of TNF-αin all 3 segments but prednisolone administration improved theinhibitory effect of 1848 (SEQ ID NO:36) (200 nmol/kg) on TNF-α levelsexclusively in the distal segment.

Example 14

Formulation of ZP1846 (SEQ ID NO:34), 10 mg/ml in histidine, mannitoland acetate formulation

-   1. Fill 800 ml (WFI) water in to a 1 L beaker-   2. Weigh 13.964 g L-histidine in a beaker and add to the 1 L beaker-   3. Weigh 32.200 g Mannitol in a beaker and add to the 1 L beaker-   4. Add 629 μL 100% acetic acid directly in to the 1 L beaker or    weigh 12.58 g of a 5% (w/v) acetic acid solution and add to the    beaker.-   6. Fill to approximately 950 ml-   7. Measure pH and adjust to pH 6.9-7.0 with 10% acetic acid or 0.25    M histidine if necessary-   8. Weigh 11.312 g Drug Substance (peptide content 88.4%) and add to    the beaker-   9. Fill to 1.015 kg (=approximately 1000 mL) and measure pH,    osmolarity and density.-   10. Sterile filter the formulation through two sterile filters    connected in series.-   11. Dispense the formulation in 0.5 mL aliquots in a LAF bench into    2 mL pharmaceutically approved vials.-   12. Partially place freeze-drying stoppers before loading into a    lyophilizer that has been sterilized and pre-cooled to 4° C.-   13. A lyophilization cycle is run over 40.5 hours consisting of    freezing, annealing, primary drying and secondary drying phases. The    vials are stoppered under nitrogen while in the lyophilizer chamber.-   14. The vials are sorted and overseals and crimps are applied.

Example 15

Formulation of ZP 1846 (SEQ ID NO:34), 10 mg/ml in histidine, arginine,mannitol and trehalose

-   1. Fill 800 ml (WFI) water in to a 1 L beaker-   2. Weigh 6.206 g L-Histidine in a beaker and add to the 1 L beaker-   3. Weigh 3.484 g L-Arginine in a beaker and add to the 1 L beaker-   4. Weigh 33.46 g Mannitol in a beaker and add to the 1 L beaker-   5. Weigh 11.16 g Trehalose in a beaker and add to the 1 L beaker-   6. Fill to approximately 950 ml-   7. Measure pH and adjust to pH 6.9-7.0 with 10% Acetic acid or 0.25    M histidine if necessary-   8. Weigh 11.312 g Drug Substance (peptide content 88.4%) and add to    the beaker-   9. Fill to 1.015 kg (=approximately 1000 ml) and measure pH,    osmolarity and density.-   10. Sterile filter the formulation through two sterile filters    connected in series.-   11. Dispense the formulation in 0.5 ml aliquots in a LAF bench into    2 ml pharmaceutically approved vials.

12. Partially place freeze-drying stoppers before loading into alyophilizer that has been sterilized and pre-cooled to 4° C.

13. A lyophilization cycle is run over 40.5 hours consisting offreezing, annealing, primary drying and secondary drying phases. Thevials are stoppered under nitrogen while in the lyophilizer chamber.

14. The vials are sorted and overseals and crimps are applied

Example 16: Method of Selecting a Patient Who is Eligible for GLP-2Analogue Therapy while Minimizing the Risk of the Patient DevelopingAdverse Effects as a Result of the Therapy

Based on the observations by Thulesen J. et al. (Gut 53:1145-1150, 2004)that both native GLP-2 and, in particular, Gly2-GLP-2 accelerate thegrowth of colonic neoplasms in mice, the present inventors have designeda selection method that aims to exclude from GLP-2 analogue therapypatients who have increased susceptibility of gastrointestinal neoplasmsor who are diagnosed of having gastrointestinal mucosal neoplasms, suchas gastric and/or colonic mucosal neoplasms.

Said method comprises permitting prescriptions for small intestineselective GLP-2 analogues, such as the analogues described by formula Iwith one or more substiutions at X3, X8, or X24 as defined in claims 15to 17 and more specifically the compounds 1846 (SEQ ID NO:34) or 1848(SEQ ID NO:36) described herein, to be prescribed by a generalpractitioner or medical specialist only after the practitioner orspecialist has obtained an approval for the prescription from a computerreadable storage medium, wherein generation of the prescription approvalcode comprises the following steps: (i) defining a plurality of patientrisk groups based upon a predefined set of risk parameters for GLP-2analogue administration; (ii) defining a set of information to beobtained from the patient, which information is probative of an adverseside effect is likely to occur if a GLP-2 analogue is administered tothe patient; (iii) in response to the information set, assigning thepatient to at least one of the risk groups and entering the patient, theinformation and the risk group assignment into the medium; (iv) basedupon the information and the risk group assignment, determining that therisk is acceptable, and, (v) upon a determination that the risk isacceptable, generating the prescription approval code to be received bythe general practitioner or specialist before the prescription isfilled.

Specifically, the definition in step (i) of various patient risk groupswould include the groups of patients already diagnosed with gastricand/or colonic mucosal cancers or the group of patients who have anincreased susceptibility to gastrointestinal mucosal cancers due to asalty diet, excess consumption of alcohol or having an otherwiseunhealthy life style; the definition of the set of information to beobtained in step (ii) would include checking for neoplasms or mucosalcell pathologies indicative of neoplastic disease, e.g. by performinggastroscopy and/or colonoscopy on the patient; and in step (iv)determining whether the risk is acceptable could also depend on theestimated duration of GLP-2 analogue therapy where the longer durationis contraindicated where certain neoplastic conditions have beenidentified in a patient.

Furthermore, the present invention has the interesting general aspect ofproviding a method of treating a small intestine disorders, such aschemotherapy induced diarrhea (CID), ulcers of the small intestine,digestion disorders and malabsorption disorders, while restrictingaccess to a GLP-2 analogue to patients with disorders related to thecolon or stomach, said method comprising permitting prescriptions forsmall intestine selective GLP-2 analogues, such as the analogues definedin claims 15 to 17, to be filled by a general practitioner or specialistonly after the practitioner or specialist has received an approval forthe prescription from a computer readable storage medium, whereingeneration of the prescription approval code comprises the followingsteps: a) defining a plurality of patient risk groups based upon apredefined set of risk parameters for said GLP-2 analogue; b) defining aset of information to be obtained by the patient, which information isprobative of an adverse side effect is likely to occur if said GLP-2analogue is administered to the patient; c) in response to theinformation set, assigning the patient to at least one of the riskgroups and entering the patient, the information and the patient's riskgroup assignment into the medium; d) based upon the information and therisk group assignment, determining that the risk is acceptable, and e)upon a determination that the risk is acceptable, generating theprescription approval code to be received by the general practitioner orspecialist before the prescription is filled.

While the invention has been described in conjunction with the exemplaryembodiments described above, many equivalent modifications andvariations will be apparent to those skilled in the art when given thisdisclosure. Accordingly, the exemplary embodiments of the invention setforth are considered to be illustrative and not limiting. Variouschanges to the described embodiments may be made without departing fromthe spirit and scope of the invention. All documents including patents,patent applications, and publications cited herein are expresslyincorporated by reference.

1. A glucagon-like peptide 2 (GLP-2) analogue represented by generalFormula I:R¹—Z¹-His-X2-X3-Gly-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-Ala-X19-X20-X21-Phe-Ile-X24-Trp-Leu-lie-X28-Thr-Lys-X31-X32-X33-Z²—R²wherein: R¹ is hydrogen, C₁₋₄ alkyl (e.g. methyl), acetyl, formyl,benzoyl or trifluoroacetyl X2 is Gly, Ala or Sar X3 is Glu or Asp X5 isSer or Thr X6 is Phe or Pro X7 is Ser or Thr X8 is Asp or Ser X9 is Gluor Asp X10 is Met, Leu, Nle or an oxidatively stable Met-replacementamino acid X11 is Asn, Ala, Lys or Ser X12 is Thr or Lys X13 is Ile, Gluor Gln X14 is Leu, Met or Nle X15 is Asp or Glu X16 is Asn or Ala X17 isLeu or Glu X18 is Ala or Aib X19 is Ala or Thr X20 is Arg or Lys X21 isAsp or Ile X24 is Asn, Ala or Glu X28 is Gln, Ala or Asn X31 is Pro, Ileor deleted X32 is Thr or deleted X33 is Asp, Asn or deleted R² is NH₂ orOH; Z¹ and Z² are independently absent or a peptide sequence of 3-20amino acid units selected from the group consisting of Ala, Leu, Ser,Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met and Orn; and whereinthe GLP-2 analogue comprises one or more of substitutions selected fromX8 is Ser and/or X16 is Ala and/or X24 is Ala and/or X28 is Ala; or apharmaceutically acceptable salt or derivative thereof.
 2. The GLP-2analogue of claim 1 which is represented by general Formula II:R¹—Z¹-His-Gly-X3-Gly-X5-Phe-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-Ala-X19-Arg-Asp-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²—R²wherein: R¹ is hydrogen, C₁₋₄ alkyl (e.g. methyl), acetyl, formyl,benzoyl or trifluoroacetyl X3 is Glu or Asp X5 is Ser or Thr X7 is Seror Thr X8 is Asp or Ser X9 is Glu or Asp X10 is Met, Leu, Nle or anoxidatively stable Met-replacement amino acid X11 is Asn, Ala, Lys orSer X12 is Thr or Lys X13 is Ile, Glu or Gln X14 is Leu, Met or Nle X15is Asp or Glu X16 is Asn or Ala X17 is Leu or Glu X19 is Ala or Thr X24is Asn or Ala X28 is Gln, Ala or Asn X31 is Pro, Ile or deleted X32 isThr or deleted X33 is Asp or deleted R² is NH₂ or OH; Z¹ and Z² areindependently absent or a peptide sequence of 3-20 amino acid unitsselected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gln,Asp, Glu, Lys, Arg, His, Met and Orn; and wherein the GLP-2 analoguecomprises one or more of substitutions selected from X8 is Ser and/orX16 is Ala and/or X24 is Ala and/or X28 is Ala; or a pharmaceuticallyacceptable salt or derivative thereof.
 3. The GLP-2 analogue of claim 1or claim 2 which is represented by general Formula III:R¹—Z¹-His-Gly-X3-Gly-X5-Phe-X7-X8-Glu-X10-X11-Thr-Ile-Leu-Asp-X16-Leu-Ala-Ala-Arg-Asp-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²—R²wherein: R¹ is hydrogen, C₁₋₄ alkyl (e.g. methyl), acetyl, formyl,benzoyl or trifluoroacetyl X3 is Glu or Asp X5 is Ser or Thr X7 is Seror Thr X8 is Asp or Ser X10 is Met, Leu, Nle, or an oxidatively stableMet-replacement amino acid X11 is Asn, Ala, Lys or Ser X24 is Asn or AlaX28 is Gln or Ala X31 is Pro or deleted X32 is Thr or deleted X33 is Aspor deleted R² is NH₂ or OH; Z¹ and Z² are independently absent or apeptide sequence of 3-20 amino acid units selected from the groupconsisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg,His, Met and Orn; and wherein the GLP-2 analogue comprises one or moreof substitutions selected from X8 is Ser and/or X16 is Ala and/or X24 isAla and/or X28 is Ala; or a pharmaceutically acceptable salt orderivative thereof.
 4. The GLP-2 analogue of any one of claims 1 to 3,wherein the GLP-2 analogue has at least 60% amino acid sequence identityto wild-type GLP-2 (1-33) and has the biological activity of causing anincrease in intestinal mass in vivo.
 5. The GLP-2 analogue of any one ofthe preceding claims, wherein the GLP-2 analogue comprises more than oneof the substitutions at positions X8, X16, X24 and/or X28 and/or one ofmore of said substitutions in combination with one or more substitutionsat positions X3, X5, X7, X10 and/or X11.
 6. The GLP-2 analogue of claim5, wherein said substitutions at position X10 is Leu, Nle, or anoxidatively stable Met-replacement amino acid, such as Met(O) orMet(O)₂.
 7. The GLP-2 analogue of claim 5, wherein said substitutions atposition X11 is Ala, Ser, or Lys.
 8. The GLP-2 analogue of claim 4,wherein the GLP-2 analogue comprises one or more of the following groupsof substitutions: Ser8, Ala16 Ser8, Ala24 Ser8, Ala28 Ala16, Ala24Ala16, Ala28 Ala24, Ala28 Ser8, Ala16, Ala24 Ser8, Ala16, Ala28 Ser8,Ala24, Ala28 Ala16, Ala24, Ala28 Ser8, Ala16, Ala24, Ala28
 9. The GLP-2analogue of claim 5, wherein the GLP-2 analogue comprises one or more ofthe following groups of substitutions: Glu3, Leu10, Ala11,24 Glu3, Thr5,Leu10, Ser11, Ala16,24,28 Glu3, Thr5, Leu10, Lys11, Ala16,24,28 Glu3,Thr5, Ser8, Leu10, Lys11, Ala16,24,28 Glu3, Thr5, Ser8,11, Leu10,Ala16,24,28 Glu3, Thr5, Ser8,11, Leu10, Ala16,24,28 Glu3, Ser8,11,Leu10, Ala16,24,28 Glu3, Leu10, Ser11, Ala16,24,28 Glu3, Leu10, Lys11,Ala16,24,28 Glu3, Thr5, Leu10, Ala11,16,24,28 Glu3, Thr5, Leu10,Ala11,16,24,28, Ile21 Glu3, Thr5, Ser8, Leu10, Ala11,16,24,28 Glu3,Ser8, Leu10, Ala11,16,24,28 Glu3, Leu10, Ala11,16,24,28 Thr7, Leu10,Ala11, 24 Thr7, Leu10, Lys11, Ala24 Thr7, Leu10, Ser11, Ala24 Thr7,Leu10, Ser8,11, Ala24 Thr7, Ser8, Leu10, Ala11,24 Thr7, Ser8, Leu10,Lys11, Ala24 Ser8, Leu10, Ala11,24 Leu10, Ala24 Leu10, Ala11, Ala24Leu10, Ala11,24,28 Leu10, Ala11,16,24,28 Leu10, Lys11, Ala24 Leu10,Ser11, Ala24 Leu10, Ser8,11, Ala24; or a deletion at one or more ofpositions X31-X33.
 10. The GLP-2 analogue of any of the preceding claimswhich is disclosed in Table 1 herein or a pharmaceutically acceptablesalt or derivative thereof.
 11. The GLP-2 analogue of claim 10 which is:1834 H-HGDGSFTSELATILDNLAARDFIAWLIQTK-NH₂ 1846H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK₆NH₂ 1847H-HGEGSFSDELSTILDALAARDFIAWLIATKITDK₆-NH₂ 1848H-HGEGTFSSELATILDALAARDFIAWLIATKITDK₆-NH₂ 1849H-HGEGSFSSELATILDALAARDFIAWLIATKITDK₆-NH₂ 1855H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH₂ 1857H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH₂ 1858H-HGEGSFSSELATILDALAARDFIAWLIATKITD-NH₂


12. The GLP-2 analogue of any one of claims 1 to 9, wherein the GLP-2analogue comprises more than one of the substitutions at positions X3,X7, X16, X24, X28, X31, X32 and/or X33.
 13. The GLP-2 analogue of claim12, wherein the GLP-2 analogue comprises one or more of substitutionsselected from X3 is Glu, X7 is Ser, X16 is Ala, X24 is Ala, X28 is Ala,X31 is Ile, X32 is Thr and X33 is Asp, and the amino acid residues inpositions X31, X32 and X33 are optionally deleted; or a pharmaceuticallyacceptable salt or derivative thereof.
 14. The GLP-2 analogue of claim12 or claim 13 which is: 1827 H-HGDGSFTDELSTILDNLAARDFIAWLIQTKKKKKKK-NH21844 H-HGEGTFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 1845H-HGEGTFSDELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 1846H-HGEGSFSSELSTILDALAARDFIAWLIATKITDKKKKKK-NH2 1848H-HGEGTFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 1849H-HGEGSFSSELATILDALAARDFIAWLIATKITDKKKKKK-NH2 1850H-HGEGSFSDELKTILDALAARDFIAWLIATKITDKKKKKK-NH2 1851H-HGEGSFSDELKTILDALAARDFIAWLIATKITDKKKKKK-NH2 1852H-HGEGTFSSELKTILDALAARDFIAWLIATKITDKKKKKK-NH2 1855H-HGEGSFSSELSTILDALAARDFIAWLIATKITD-NH2 1857H-HGEGTFSSELATILDALAARDFIAWLIATKITD-NH2 1858H-HGEGSFSSELATILDALAARDFIAWLIATKITD-NH2 1859H-HGEGSFSDELKTILDALAARDFIAWLIATKITD-NH2;

or a pharmaceutically acceptable salt or derivative thereof.
 15. TheGLP-2 analogue of any one of claims 1 to 9, wherein the GLP-2 analoguecomprises more than one of the substitutions at positions X3, X8 and/orX24.
 16. The GLP-2 analogue of claim 15, wherein the GLP-2 analoguecomprises one or more of substitutions selected from X3 is Asp, X8 isAsp and X24 is Ala; and the amino acid residues in positions X31, X32and X33 are optionally deleted; or a pharmaceutically acceptable salt orderivative thereof.
 17. The GLP-2 analogue of claim 15 or claim 16 whichis: 1830 H-HGDGSFSDELSTILDNLAARDFIAWLIQTK-NH2 1831H-HGDGSFTDELSTILDNLAARDFIAWLIQTK-NH2 1835H-HGDGSFSDELKTILDNLAARDFIAWLIQTK-NH2 1836H-HGDGSFTDELKTILDNLAARDFIAWLIQTK-NH2 1839H-HGDGSFSDELATILDNLAARDFIAWLIQTKITDKKKKKK-NH2 1840H-HGDGSFSDELATILDNLAARDFIAWLIQTKITD-NH2 1841H-HGDGSFSDELATILDNLAARDFIAWLIQTK-NH2 1843H-HGDGSFTDELATILDNLAARDFIAWLIQTK-NH2;

or a pharmaceutically acceptable salt or derivative thereof.
 18. TheGLP-2 analogue of any one of claims 1 to 9 which possesses asubstitution at one or more of positions X3, X33, X10, X11, X16 and/orX24.
 19. A GLP-2 analogue of any one of the preceding claims for use intherapy.
 20. A pharmaceutical composition comprising a GLP-2 analogue ofany one of the preceding claims, or a salt or derivative thereof, inadmixture with a carrier.
 21. The pharmaceutical composition of claim20, wherein the GLP-2 analogue is a pharmaceutically acceptable acidaddition salt.
 22. The pharmaceutical composition of claim 20 or claim21, which is formulated as a liquid suitable for administration byinjection or infusion, or which is formulated to cause slow release ofsaid GLP-2 analogue.
 23. Use of a GLP-2 analogue of any one of claims 1to 18 for the preparation of a medicament for the treatment and/orprevention of a stomach and bowel-related disorder.
 24. The use of claim23, wherein the stomach and bowel-related disorder is ulcers, digestiondisorders, malabsorption syndromes, short-gut syndrome, cul-de-sacsyndrome, inflammatory bowel disease, celiac sprue (for example arisingfrom gluten induced enteropathy or celiac disease), tropical sprue,hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn'sdisease), ulcerative colitis, small intestine damage or short bowelsyndrome.
 25. The use of claim 23, wherein the stomach and bowel-relateddisorder is radiation enteritis, infectious or post-infectiousenteritis, or small intestinal damage due to toxic or otherchemotherapeutic agents.
 26. Use of a GLP-2 analogue of any one ofclaims 1 to 18 for the preparation of a medicament for the treatmentand/or prevention of a side effect of chemotherapy or radiationtreatment.
 27. The use of claim 26, wherein the side effect ofchemotherapy is diarrhoea, abdominal cramping, vomiting or structuraland functional damage of the intestinal epithelium resulting fromchemotherapy treatment.
 28. Use of a GLP-2 analogue of any one of claims1 to 18 for the preparation of a medicament for the treatment ofneo-natals, osteoporosis or DPP-IV (dipeptidylpeptidase-IV) mediatedconditions.
 29. Use of a GLP-2 analogue of any one of claims 1 to 18 forthe preparation of a medicament for the treatment and/or prevention of acondition involving malnutrition.
 30. The use of claim 29, wherein thecondition involving malnutrition is cachexia or anorexia.
 31. A nucleicacid molecule comprising a nucleic acid sequence encoding a GLP-2analogue of any one of claims 1 to
 18. 32. An expression vectorcomprising the nucleic acid sequence of claim 31, in combination withcontrol sequences to directed its expression.
 33. A host celltransformed the expression vector of claim
 32. 34. A method of producingthe GLP-2 analogue of any one of claims 1 to 18, the method comprisingculturing the host cells of claim 22 under conditions suitable forexpressing the GLP-2 analogue and purifying the GLP-2 analogue thusproduced.
 35. A nucleic acid molecule according to claim 31, anexpression vector according to claim 32, or a host cell according toclaim 33, for use in therapy.
 36. Use of a nucleic acid moleculeaccording to claim 31, an expression vector according to claim 32, or ahost cell according to claim 33, in the preparation of a medicament forthe treatment and/or prevention of a stomach and bowel-related disorder,or for the treatment and/or prevention of a side effect of chemotherapyor radiation treatment, or for the treatment of neo-natals, osteoporosisor DPP-IV (dipeptidylpeptidase-IV) mediated conditions.
 37. A method oftreating a stomach and bowel-related disorder in a patient in needthereof by administering an effective amount a GLP-2 analogue of any oneof claims 1 to 17, a nucleic acid molecule according to claim 31, anexpression vector according to claim 32, or a host cell according toclaim
 33. 38. The method of claim 37, wherein the stomach andbowel-related disorder is ulcers, digestion disorders, malabsorptionsyndromes, short-gut syndrome, cul-de-sac syndrome, inflammatory boweldisease, celiac sprue (for example arising from gluten inducedenteropathy or celiac disease), tropical sprue, hypogammaglobulinemicsprue, enteritis, regional enteritis (Crohn's disease), ulcerativecolitis, small intestine damage or short bowel syndrome.
 39. The methodof claim 37, wherein the stomach and bowel-related disorder is radiationenteritis, infectious or post-infectious enteritis, or small intestinaldamage due to toxic or other chemotherapeutic agents.
 40. A method oftreating or preventing a side effect of chemotherapy or radiationtherapy to a patient in need thereof, the method comprisingadministering an effective amount a GLP-2 analogue of any one of claims1 to 18, a nucleic acid molecule according to claim 31, an expressionvector according to claim 32, or a host cell according to claim
 33. 41.The method of claim 40, wherein the side effect of chemotherapy isdiarrhoea, abdominal cramping, vomiting or structural or functionaldamage of the intestinal epithelium resulting from chemotherapytreatment.
 42. A method of treating neo-natal disorders, obesity,osteoporosis or DPP-IV (dipeptidylpeptidase-IV) mediated conditions in apatient in need thereof, the method comprising administering aneffective amount a GLP-2 analogue of any one of claims 1 to 18, anucleic acid molecule according to claim 31, an expression vectoraccording to claim 32, or a host cell according to claim
 33. 43. Atherapeutic kit comprising a cancer chemotherapy drug and a GLP-2analogue according to any one of claims 1 to 18, a nucleic acid moleculeaccording to claim 31, an expression vector according to claim 32, or ahost cell according to claim 33, each optionally in combination with apharmaceutically acceptable carrier.
 44. A pharmaceutical compositioncomprising a cancer chemotherapy drug and a GLP-2 analogue according toany one of claims 1 to 18, a nucleic acid molecule according to claim31, an expression vector according to claim 32, or a host cell accordingto claim 33 in combination with a pharmaceutically acceptable carrier.45. A GLP-2 analogue of any of claims 1 to 17 wherein said analog hasenhanced stability relative to Gly2-GLP-2.
 46. A GLP-2 analogue of anyof claims 1 to 17 which is further characterized in having an observedpurity of at least 70% relative to the initial purity in conditionsselected from the group consisting of 0.1 M HCl after 12 days, 0.5% H₂O₂after 3 days, and 0.1 M NH₄HCO₃ after 6 days
 47. A GLP-2 analogue of anyof claims 1 to 17 which is further characterized in having an observedpurity of at least 60% relative to initial purity in a solution of HCl0.1 M after 12 days.
 48. A GLP-2 analogue of any of claims 1 to 17 whichis further characterized in having an observed purity of at least 70%relative to initial purity in a solution of NH₄HCO₃ 0.1 M after 6 days.